Associations of microbiota and toll-like receptor signaling pathway in esophageal adenocarcinoma.

BACKGROUND: Toll-like receptors (TLRs) recognize known molecules from microbes and have an established role in tumorigenesis. Using a rat model of esophageal adenocarcinoma, and human clinical samples, we investigated genes central to TLR-mediated signal transduction and characterized the esophageal microbiome across the spectrum of esophageal adenocarcinoma carcinogenesis. METHODS: We surgically induced bile/acid reflux in rats and their esophagi were harvested at 40 weeks post-surgery. Tissue samples from the model were selected for gene expression profiling. Additionally, for rat and human samples microbiome analysis was performed using PCR-ESI-MS-TOF technology with validation by fluorescence in situ hybridization. RESULTS: Gene expression results in the rat model indicated a significant upregulation of TLRs 1-3, 6, 7 and 9 in EAC compared to normal epithelium. PCR-ESI-MS-TOF analysis revealed a prevalence of Escherichia coli in Barrett's esophagus (60%) and esophageal adenocarcinoma (100%), which was validated by fluorescence in situ hybridization. In the human clinical samples, Streptococcus pneumonia was detected in high abundance in gastroesophageal reflux disease and Barrett's esophagus (50-70%) in comparison to tumor adjacent normal epithelium, dysplasia, and esophageal adenocarcinoma (20-30%). E. coli was detected in the Barrett's esophagus and esophageal adenocarcinoma groups but was absent in the tumor adjacent normal epithelium, dysplasia, and the gastroesophageal reflux disease groups. CONCLUSIONS: We demonstrated an association between the TLR signaling pathway and E. coli hinting towards possible early molecular changes being mediated by microbes in the rat model of esophageal adenocarcinoma carcinogenesis. Studies on human clinical samples also corroborate results to some extent; however, a study with larger sample size is needed to further explore this association.

Multifunctional role of human SPLUNC1 in Pseudomonas aeruginosa infection.

The human short PLUNC1 (SPLUNC1) protein has been identified as a component of the pulmonary antimicrobial response based on its structural similarity to the bactericidal/permeability-increasing (BPI) protein. Using a genetically modified mouse model, we recently verified the antimicrobial activity of SPLUNC1 against Pseudomonas aeruginosa in vivo. To further define the mechanism of epithelial SPLUNC1-mediated antibacterial action, we carried out studies to determine how SPLUNC1 protects the host from acute respiratory infections. P. aeruginosa treated with recombinant human SPLUNC1 protein showed decreased growth in vitro. This antibacterial activity was due to growth inhibition, as a consequence of a SPLUNC1-induced increase in bacterial cell permeability. Removal of SPLUNC1 allowed the recovery of P. aeruginosa and suggested no permanent cell injury or direct killing of bacteria. Further investigation showed coating of bacterial cells by SPLUNC1. We suggest that this "bacterial cell coating" is necessary for the bacteriostatic function of SPLUNC1. Additionally, we demonstrated a novel role for SPLUNC1 as a chemoattractant that facilitated migration of macrophages and neutrophils. Taking the findings together, we propose synergistic roles for human SPLUNC1 as an antibacterial agent with bacteriostatic and chemotactic activities.

The Presence of Biofilms in Instrumented Spinal Fusions.

Study Design: Prospective observational cohort study. Objective: To determine whether biofilms exist on spinal instrumentation recovered during revision surgery in which microbial cultures were negative. Background: Biofilm bacteria are extremely difficult to detect by conventional culture methods used in the standard hospital setting. Chronic infections in which bacteria form biofilms have been demonstrated to slow healing and prevent bony fusion. These slime encased microbial communities serve to isolate the bacteria from the body's immune responses, while simultaneously providing metabolic resistance to antimicrobial therapy. Methods: Traditional debridement wound cultures were taken from each specimen and sent for microbiological analyses. Bacterial DNA testing was performed using polymerase chain reaction (PCR) electrospray ionization-mass spectrometry (ESI-MS). Based on the PCR/ESI-MS results, specific crossed immune electrophoresis was used to detect the bacterial species within biofilms observed on the removed instrumentation. In addition, fluorescent in situ hybridization (FISH) probes corresponding to the bacterial species identified by PCR/ESI-MS were used with confocal microscopy to visualize and confirm the infecting bacteria. Results: Fifteen patients presented for surgical revision of thoracolumbar spinal implantation: four for clinical suspicion of infection, six for adjacent segment disease (ASD), one with ASD and pseudoarthrosis (PA), three with PA, and one for pain. Infections were confirmed with PCR/ESI-MS for all four patients who presented with clinical infection, and for five of the patients for whom infection was not clinically suspected. Of the presumed non-infected implants, 50% demonstrated the presence of infectious biofilms. Half of the revisions due to pseudoarthrosis were shown to harbour biofilms. The revisions that were performed for pain demonstrated robust biofilms but did not grow bacteria on traditional culture media. Conclusions: Culture is inadequate as a diagnostic modality to detect indolent/subclinical biofilm infections of spinal instrumentation. The PCR/ESI-MS results for bacterial detection were confirmed using species-specific microscopic techniques for both bacterial nucleic acids and antigens. Biofilms may contribute to pseudoarthrosis and back pain in postoperative wounds otherwise considered sterile.

Bacterial Identification and Visualization of Bacterial Biofilms Adjacent to Fracture Sites After Internal Fixation.

Objectives: The primary aims of this study were to determine if any correlation exists in cases of fracture fixation among: (1) bacterial profiles recovered from the instrumentation and adjacent tissues; (2) the type of orthopedic injury; and (3) the clinical outcome-union versus nonunion. A secondary goal was to compare culture and molecular diagnostics for identifying the bacterial species present following fracture fixation. Design: Single-institution, prospective case-control cohort study. Setting: Single level 1 trauma center. Patients: Forty-nine bony nonunion cases undergoing revision internal fixation and 45 healed fracture controls undergoing removal of hardware. Intervention: Bacterial infection was detected by standard microbial culture methods and by a pan-eubacterial domain, molecular diagnostic (MDx) assay. Confirmation of culture and MDx results was achieved with bacterial ribosomal 16S rRNA fluorescence in situ hybridization (FISH) to visualize bacterial biofilms. Main Outcome Measurements: MDx and microbial culture methods results were the primary study outcomes. Results: Ninety-four percent of the nonunion cohort and 93% of the union cohort had bacteria detected by the MDx. Seventy-eight percent of the nonunion cases and 69% of the controls were culture negative, but MDx positive. Although no significant differences in bacterial composition were observed between the cases and controls, differences were observed when cases were divided by comorbidities. Conclusion: The MDx is more sensitive than microbial culture in detecting bacterial presence. The lack of significantly different findings with regard to bacterial profile identified between the cases and controls suggests that host factors and environmental conditions are largely responsible for determining if bony union will occur. Level of Evidence: Diagnostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Laser disruption and killing of methicillin-resistant Staphylococcus aureus biofilms.

OBJECTIVE: The aim of the study was to study the efficacy of 2 different lasers in vitro, in disrupting biofilm and killing planktonic pathogenic bacteria. MATERIALS AND METHODS: Biofilms of a stable bioluminescent of Staphylococcus aureus Xen 31 were grown in a 96-well microtiter plate for 3 days. The study included 7 arms: (a) control; (b) ciprofloxacin (3 mg/L, the established minimum inhibitory concentration [MIC]) alone; (c) shock wave (SW) laser alone; (d) near-infrared (NIR) laser alone; (e) SW laser and ciprofloxacin; (f) SW and NIR lasers; (g) SW, NIR lasers, and ciprofloxacin. The results were evaluated with an in vivo imaging system (IVIS) biophotonic system (for live bacteria) and optical density (OD) for total bacteria. RESULTS: Without antibiotics, there was a 43% reduction in OD (P < .05) caused by the combination of SW and NIR suggesting that biofilm had been disrupted. There was an 88% reduction (P < .05) in live biofilm. Ciprofloxacin alone resulted in a decrease of 28% of total live cells (biofilm remaining attached) and 58% of biofilm cells (both P > .05). Ciprofloxacin in combination with SW and SW + NIR lasers caused a decrease of more than 60% in total live biomass and more than 80% of biofilm cells, which was significantly greater than ciprofloxacin alone (P < .05). CONCLUSIONS: We have demonstrated an effective nonpharmacologic treatment method for methicillin-resistant Staphylococcus aureus (MRSA) biofilm disruption and killing using 2 different lasers. The preferred treatment sequence is a SW laser disruption of biofilm followed by NIR laser illumination. Treatment optimization of biofilm is possible with the addition of ciprofloxacin in concentrations consistent with planktonic MIC.

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

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 mum) 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.

Direct demonstration of Staphylococcus biofilm in an external ventricular drain in a patient with a history of recurrent ventriculoperitoneal shunt failure.

External ventricular drains (EVD) are associated with a high infection rate. Early detection of infection is frequently problematic due to a lack of clinical signs and the time period required for culturing. Bacterial biofilms have been suggested to play an important role in the infection of EVD, but direct evidence is as yet lacking. We report the case of a 17- year-old male with Dandy-Walker malformation who presented with headache, nausea and drowsiness; a CT scan revealed enlarged ventricles. The patient had a history of ventriculoperitoneal shunt revision 3 weeks prior to admission. The shunt was removed on suspicion of infection and an EVD placed. Daily surveillance cultures through the EVD were negative and the EVD was replaced on day 5. Examination of the initial EVD by confocal microscopy demonstrated clear intraluminal biofilm formation; molecular analysis by PCR identified Staphylococcus aureus resident on the catheter. To our knowledge, this is the first direct demonstration of an intraluminal biofilm compromising an EVD. Despite the presence of biofilm on this catheter, the patient demonstrated no clinical signs of infection, and the routine surveillance culture was negative. Undetected biofilm may pose a latent risk on EVD and other neurosurgical catheters.

Fluorescence "in situ" hybridization for the detection of biofilm in the middle ear and upper respiratory tract mucosa.

Most chronic bacterial infections are associated with biofilm formation wherein the bacteria attach to mucosal surfaces, wound tissue, or medical device surfaces in the human body via the formation of an extracellular matrix. Biofilms assume complex three-dimensional structures dependent on the species, the strain, and the prevailing environmental conditions and are composed of both the bacteria and the extracellular slime-like matrices, which surround the bacteria. Bacteria deep in the biofilm live under anaerobic conditions and must use alternatives to O(2) as a terminal electron acceptor. Thus, the metabolic rates of these deep bacteria are greatly reduced, which renders them extremely resistant to antibiotic treatment, and for reasons not clearly understood, it is often very difficult to culture biofilm bacteria using traditional microbiologic techniques. To directly identify and visualize biofilm bacteria in a species-specific manner, we developed a confocal laser scanning microscopy (CLSM)-based 16S rRNA fluorescence in situ hybridization (FISH) protocol, to find biofilm bacteria in middle ear and upper respiratory tract mucosa, which preserves the three-dimensional structure of the biofilm and avoids the use of traditional culture techniques.

Tonsillolith: not just a stone but a living biofilm.

OBJECTIVE: To study the morphology and activity of tonsilloliths, demonstrating oxygen respiration, denitrification, and acidification on exposure to sucrose. STUDY DESIGN: Tonsilloliths were extracted in atraumatic conditions during tonsillectomy from 16 adults and sent to two different laboratories for histological, bacteriological, and biofilm studies under sterile conditions. SETTING: Multicenter laboratory study. SUBJECTS AND METHODS: Multiple tonsilloliths from two patients examined by confocal microscopy and microelectrodes were used to measure aerobic/anaerobic respiration and acid production (dissolved oxygen, nitrous oxide, pH) when exposed to saliva following addition of sucrose and fluoride. RESULTS: Morphologically, tonsilloliths were similar to dental biofilms, containing corncob structures, filaments, and cocci. Microelectrodes showed that the microorganisms respired oxygen and nitrate. The oxygen concentration in the center of the tonsillolith was depleted to approximately one-tenth of that of the overlying fluid. The addition of sucrose resulted in acid production within the tonsillolith, dropping the pH from 7.3 to 5.8. The data showed stratification with oxygen respiration at the outer layer of tonsillolith, denitrification toward the middle, and acidification toward the bottom. The depletion of oxygen and acid production following addition of sucrose may allow the proliferation of anaerobic/acidophilic bacteria. Fluoride suppressed acid production in the presence of sucrose. CONCLUSIONS: Tonsilloliths exhibit biofilm structure and the formation of chemical gradients through physiological activity. Although tonsillectomy is an option for treating cryptic infections, understanding the morphology and biofilm characteristics of tonsilloliths may stimulate scientists to use limited or targeted remedies in the future.

Resistance of Synthetic and Biologic Surgical Meshes to Methicillin-Resistant Staphylococcus aureus Biofilm: An In Vitro Investigation.

Surgical meshes have become the standard procedure for a variety of surgical applications with 20 million meshes being implanted each year. The popularity of mesh usage among surgeons is backed by the multiple studies that support its functionality as a tool for improving surgical outcomes. However, their use has also been associated with infectious surgical complications and many surgeons have turned to biologic meshes. While there have been several studies investigating synthetic meshes, there is limited data comparing synthetic and biologic meshes in vitro in an infection model. This study evaluates the in vitro susceptibility of both synthetic and biologic meshes to single-species methicillin-resistant Staphylococcus aureus (MRSA) biofilms. This research compares biofilm biomass, average thickness, and coverage between the three meshes through florescent in situ hybridization (FISH), confocal scanning microscopy (CSLM), and image analysis. We also report the varying levels of planktonic and attached bacteria through sonication and cfu counts. While the data illustrates increased biofilm formation on biologic mesh in vitro, the study must further be investigated in vivo to confirm the study observations.

Evaluation of bacterial presence on lead X-ray aprons utilised in the operating room via IBIS and standard culture methods.

BACKGROUND: Despite precautions, surgical procedures carry risk of infection. Radiation-protective lead aprons worn by operating personnel are a potential source of bacterial contamination and have not been fully evaluated. AIM/OBJECTIVE: To evaluate lead aprons as a source of bacterial contamination, identify organisms most commonly found on this source, and devise a method with which to lower the risk of contamination. METHODS: In this basic science study, 20 randomly selected lead X-ray aprons were swabbed at three time points. The experimental treatment was with a hospital-grade disinfectant wipe. The samples were assessed for bacterial growth via traditional plating methods and mass spectrometry. Plates were graded on a scale of 0 to 4+ based on the number of quadrants with growth. Growth on one quadrant or more was considered contaminated. FINDINGS/RESULTS: Bacteria were initially detected via IBIS on a majority of the aprons (32/40), most commonly Staphylococcus epidermidis and Propionibacterium acnes. Virulent organisms cultured were Methicillin-resistant Staphylococcus epidermidis (MRSE), Neisseria, Streptococcus viridans and pseudomonas. MRSE were detected on 5/20 of the samples. Immediately after treatment, the majority of aprons showed less bacterial contamination (0/20 standard culture positive; 13/20 IBIS positive) with some recurrence at the 6-h time point (2/20 standard culture positive, 16/20 IBIS positive). All MRSE detected initially was eradicated. DISCUSSION: Lead X-ray aprons worn in the operating room harbour bacteria. Disinfecting before use may prevent the introduction of virulent organisms to patients. Our proposed method of sanitising with a disinfectant wipe is quick and effective.

Silver-Coated Endotracheal Tubes Cleaned With a Mechanism for Secretion Removal.

BACKGROUND: Biofilm on the surface of endotracheal tubes (ETTs) is associated with ventilator-associated pneumonia. The use of silver-coated ETTs has been suggested to reduce the occurrence of ventilator-associated pneumonia by preventing biofilm formation. However, mucus accumulation can reduce the antibacterial activity of silver-coated ETTs by isolating bacterial colonies from the silver surface. We hypothesized that, in mechanically ventilated subjects, periodic removal of secretions through the use of a cleaning device would enhance the antimicrobial properties of silver-coated ETTs and thus reduce bacterial colonization. METHODS: Subjects were randomized to either standard suctioning (blind tracheal suctioning, control group) or blind tracheal suctioning plus cleaning maneuver every 8 h (treatment group). Tracheal aspirates were collected immediately before extubation for microbiological culture. After extubation, ETTs were collected for both cultural and non-cultural microbiological analysis and biofilm isolation. RESULTS: 39 subjects expected to be ventilated for > 48 h were enrolled; 36 ETTs (18 control, 18 treatment) and 29 tracheal samples (15 control, 14 treatment) were collected. Among the ETTs positive for bacterial colonization (15 vs 9, P = .18), cleaning maneuvers did not reduce microbial load, shown as the decimal logarithm of colony-forming units (CFU) per mL (1.6 ± 1.2 vs 0.9 ± 1.2 logCFU/mL, P = .15). There was a trend toward decreased biofilm deposition (439.5 ± 29.0 vs 288.9 ± 157.7 mg, P = .09) in the treated ETTs. No significant differences were observed in the number of positive tracheal aspirates (13 vs 10, P = .39) or in the microbial load (4.8 ± 4.0 vs 4.2 ± 3.8 logCFU/mL, P = .70) of tracheal secretions. Finally, no differences in the microbial load of Gram-positive organisms, Gram-negative organisms, or yeasts were found between the ETTs and tracheal aspirates of the 2 groups. CONCLUSIONS: In 39 critically-ill subjects intubated with silver-coated ETTs, periodic cleaning maneuvers did not decrease bacterial colonization of the ETTs and did not lower respiratory tract colonization compared to the standard suctioning. (Clinicaltrials.gov registration NCT02120001.).

Deletion of genes involved in the ketogluconate metabolism, Entner-Doudoroff pathway, and glucose dehydrogenase increase local and invasive virulence phenotypes in Streptococcus pneumoniae.

Streptococcus pneumoniae displays increased resistance to antibiotic therapy following biofilm formation. A genome-wide search revealed that SP 0320 and SP 0675 (respectively annotated as 5-keto-D-gluconate-5-reductase and glucose dehydrogenase) contain the highest degree of homology to CsgA of Myxococcus xanthus, a signaling factor that promotes cell aggregation and biofilm formation. Single and double SP 0320 and SP 0675 knockout mutants were created in strain BS72; however, no differences were observed in the biofilm-forming phenotypes of mutants compared to the wild type strain. Using the chinchilla model of otitis media and invasive disease, all three mutants exhibited greatly increased virulence compared to the wild type strain (increased pus formation, tympanic membrane rupture, mortality rates). The SP 0320 gene is located in an operon with SP 0317, SP 0318 and SP 0319, which we bioinformatically annotated as being part of the Entner-Doudoroff pathway. Deletion of SP 0317 also resulted in increased mortality in chinchillas; however, mutations in SP 0318 and SP 0319 did not alter the virulence of bacteria compared to the wild type strain. Complementing the SP 0317, SP 0320 and SP 0675 mutant strains reversed the virulence phenotype. We prepared recombinant SP 0317, SP 0318, SP 0320 and SP 0675 proteins and confirmed their functions. These data reveal that disruption of genes involved in the degradation of ketogluconate, the Entner-Doudoroff pathway, and glucose dehydrogenase significantly increase the virulence of bacteria in vivo; two hypothetical models involving virulence triggered by reduced in carbon-flux through the glycolytic pathways are presented.

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

BACKGROUND: Streptococcus 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. RESULTS: Multiparametric 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. CONCLUSION: All 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 REVIEW: Biofilms 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 FINDINGS: The 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. SUMMARY: The 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 detection of bacterial biofilms on the middle-ear mucosa of children with chronic otitis media.

CONTEXT: Chronic otitis media (OM) is a common pediatric infectious disease. Previous studies demonstrating that metabolically active bacteria exist in culture-negative pediatric middle-ear effusions and that experimental infection with Haemophilus influenzae in the chinchilla model of otitis media results in the formation of adherent mucosal biofilms suggest that chronic OM may result from a mucosal biofilm infection. OBJECTIVE: To test the hypothesis that chronic OM in humans is biofilm-related. DESIGN, SETTING, AND PATIENTS: Middle-ear mucosa (MEM) biopsy specimens were obtained from 26 children (mean age, 2.5 [range, 0.5-14] years) undergoing tympanostomy tube placement for treatment of otitis media with effusion (OME) and recurrent OM and were analyzed using microbiological culture, polymerase chain reaction (PCR)-based diagnostics, direct microscopic examination, fluorescence in situ hybridization, and immunostaining. Uninfected (control) MEM specimens were obtained from 3 children and 5 adults undergoing cochlear implantation. Patients were enrolled between February 2004 and April 2005 from a single US tertiary referral otolaryngology practice. MAIN OUTCOME MEASURES: Confocal laser scanning microscopic (CLSM) images were obtained from MEM biopsy specimens and were evaluated for biofilm morphology using generic stains and species-specific probes for H influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis. Effusions, when present, were evaluated by PCR and culture for evidence of pathogen-specific nucleic acid sequences and bacterial growth, respectively. RESULTS: Of the 26 children undergoing tympanostomy tube placement, 13 (50%) had OME, 20 (77%) had recurrent OM, and 7 (27%) had both diagnoses; 27 of 52 (52%) of the ears had effusions, 24 of 24 effusions were PCR-positive for at least 1 OM pathogen, and 6 (22%) of 27 effusions were culture-positive for any pathogen. Mucosal biofilms were visualized by CLSM on 46 (92%) of 50 MEM specimens from children with OME and recurrent OM using generic and pathogen-specific probes. Biofilms were not observed on 8 control MEM specimens obtained from the patients undergoing cochlear implantation. CONCLUSION: Direct detection of biofilms on MEM biopsy specimens from children with OME and recurrent OM supports the hypothesis that these chronic middle-ear disorders are biofilm-related.

A novel technique using potassium permanganate and reflectance confocal microscopy to image biofilm extracellular polymeric matrix reveals non-eDNA networks in Pseudomonas aeruginosa biofilms.

Biofilms are etiologically important in the development of chronic medical and dental infections. The biofilm extracellular polymeric substance (EPS) determines biofilm structure and allows bacteria in biofilms to adapt to changes in mechanical loads such as fluid shear. However, EPS components are difficult to visualize microscopically because of their low density and molecular complexity. Here, we tested potassium permanganate, KMnO4, for use as a non-specific EPS contrast-enhancing stain using confocal laser scanning microscopy in reflectance mode. We demonstrate that KMnO4 reacted with EPS components of various strains of Pseudomonas, Staphylococcus and Streptococcus, yielding brown MnO2 precipitate deposition on the EPS, which was quantifiable using data from the laser reflection detector. Furthermore, the MnO2 signal could be quantified in combination with fluorescent nucleic acid staining. COMSTAT image analysis indicated that KMnO4 staining increased the estimated biovolume over that determined by nucleic acid staining alone for all strains tested, and revealed non-eDNA EPS networks in Pseudomonas aeruginosa biofilm. In vitro and in vivo testing indicated that KMnO4 reacted with poly-N-acetylglucosamine and Pseudomonas Pel polysaccharide, but did not react strongly with DNA or alginate. KMnO4 staining may have application as a research tool and for diagnostic potential for biofilms in clinical samples.

Polymerase Chain Reaction-Electrospray-Time-of-Flight Mass Spectrometry Versus Culture for Bacterial Detection in Septic Arthritis and Osteoarthritis.

BACKGROUND: Preliminary studies have identified known bacterial pathogens in the knees of patients with osteoarthritis (OA) before arthroplasty. AIMS: The current study was designed to determine the incidence and types of bacteria present in the synovial fluid of native knee joints from adult patients with diagnoses of septic arthritis and OA. PATIENTS AND METHODS: Patients were enrolled between October 2010 and January 2013. Synovial fluid samples from the affected knee were collected and evaluated with both traditional microbial culture and polymerase chain reaction-electrospray ionization-time-of-flight mass spectrometry (molecular diagnostics [MDx]) to prospectively characterize the microbial content. Patients were grouped by diagnosis into one of two cohorts, those with clinical suspicion of septic arthritis (n = 44) and those undergoing primary arthroplasty of the knee for OA (n = 21). In all cases where discrepant culture and MDx results were obtained, we performed species-specific 16S rRNA fluorescence in situ hybridization (FISH) as a confirmatory test. RESULTS: MDx testing identified bacteria in 50% of the suspected septic arthritis cases and 29% of the arthroplasty cases, whereas culture detected bacteria in only 16% of the former and 0% of the latter group. The overall difference in detection rates for culture and MDx was very highly significant, p-value = 2.384 × 10-7. All of the culture-positive cases were typed as Staphylococcus aureus. Two of the septic arthritis cases were polymicrobial as was one of the OA cases by MDx. FISH testing of the specimens with discordant results supported the MDx findings in 91% (19/21) of the cases, including one case where culture detected S. aureus and MDx detected Streptococcus agalactiae. CONCLUSIONS: MDx were more sensitive than culture, as confirmed by FISH. FISH only identifies bacteria that are embedded or infiltrated within the tissue and is thus not susceptible to contamination. Not all suspected cases of septic arthritis contain bacteria, but a significant percent of patients with OA, and no signs of infection, have FISH-confirmed bacterial biofilms present in the knee.

Direct demonstration of bacterial biofilms on prosthetic mesh after ventral herniorrhaphy.

BACKGROUND: Prosthetic mesh is employed routinely in the treatment of ventral and parastomal hernias, but its use can lead to major complications, including infection, extrusion, and fistula. Bacterial biofilms have been posited to play a role in mesh-related infection, but although bacteria have been noted to form biofilms on mesh surfaces in vitro, they have never been visualized directly in biofilms on mesh recovered from patients experiencing infectious complications. METHODS: Five patients who developed complications after ventral hernia repair with prosthetic mesh were operated on again. Explanted mesh was examined for biofilm with confocal laser scanning microscopy (CLSM) and fluorescence in situ hybridization (FISH). In two cases, a novel molecular assay (the Ibis T5000) was used to characterize the biofilm-forming bacteria. RESULTS: The CLSM examination demonstrated adherent biofilms on mesh surfaces in all five patients. Biofilms also were noted on investing fibrous tissue. The FISH study was able to discriminate between bacterial species in polymicrobial biofilms. In two patients the Ibis T5000 detected more species of constituent biofilm bacteria than did standard culture. Removal of the mesh and reconstruction with autologous tissues or biologic materials resolved the presenting complaints in all cases. CONCLUSION: Bacterial biofilms should be considered an important contributor to the pathology and complications associated with prosthetic mesh implanted in the abdominal wall. If biofilms are present, complete removal of the mesh and repair of the resulting defect without alloplastic materials is an effective intervention.

Presence of bacteria in failed anterior cruciate ligament reconstructions.

BACKGROUND: Novel microbial detection technologies have revealed that chronic bacterial biofilms, which are recalcitrant to antibiotic treatment, are common in failed orthopedic procedures. QUESTIONS: Are bacteria present on failed anterior cruciate ligament (ACL) reconstructions? Is there a difference in the presence or nature of bacteria in failed ACL reconstructions relative to a control set of healthy ACL's? METHODS: We used a case-control study design, where we analyzed the bacterial composition of 10 failed ACL reconstructions and compared it to 10 native ACL's harvested during total knee arthroplasty. The IBIS Universal Biosensor was used to determine the nature of bacteria on ACL specimens, and fluorescent in situ hybridization (FISH) was used to visualize bacteria in a subset of cases. RESULTS: Bacteria are present in failed ACL reconstructions. Bacteria are present in ACL's harvested during total knee arthroplasty, but the nature of the species differs significantly between experimental and control sets. Twelve genera were detected in the experimental set (in both allografts and autografts), and in four samples multiple species were detected. In contrast, the control group was characterized by presence of Propionibacterium acnes. CONCLUSIONS: We demonstrate the presence of bacteria on failed ACLs surgeries, and open the door to investigate whether and how bacteria and the associated immune responses could possibly contribute to graft failure. CLINICAL RELEVANCE: If microbial pathogens can be linked to failed grafts, it could provide: (1) markers for early diagnosis of abnormal healing in ACL surgeries, and (2) targets for early treatment to prevent additional reconstruction surgeries.