AbstractObjectivesThe cephalosporin nitric oxide (NO)-donor prodrug DEA-C3D (‘DiEthylAmin-Cephalosporin-3′-Diazeniumdiolate’) has been shown to initiate the dispersal of biofilms formed by the Pseudomonas aeruginosa laboratory strain PAO1. In this study, we investigated whether DEA-C3D disperses biofilms formed by clinical cystic fibrosis (CF) isolates of P. aeruginosa and its effect in combination with two antipseudomonal antibiotics, tobramycin and colistin, in vitro.Methodsβ-Lactamase-triggered release of NO from DEA-C3D was confirmed using a gas-phase chemiluminescence detector. MICs for P. aeruginosa clinical isolates were determined using the broth microdilution method. A crystal violet staining technique and confocal laser scanning microscopy were used to evaluate the effects of DEA-C3D on P. aeruginosa biofilms alone and in combination with tobramycin and colistin.ResultsDEA-C3D was confirmed to selectively release NO in response to contact with bacterial β-lactamase. Despite lacking direct, cephalosporin/β-lactam-based antibacterial activity, DEA-C3D was able to disperse biofilms formed by three P. aeruginosa clinical isolates. Confocal microscopy revealed that DEA-C3D in combination with tobramycin produces similar reductions in biofilm to DEA-C3D alone, whereas the combination with colistin causes near complete eradication of P. aeruginosa biofilms in vitro.ConclusionsDEA-C3D is effective in dispersing biofilms formed by multiple clinical isolates of P. aeruginosa and could hold promise as a new adjunctive therapy to patients with CF.

Non-surface attached bacterial aggregates are frequently found in clinical settings associated with chronic infections. Current methods quantifying the extent to which a suspended bacterial population is aggregated mainly rely on: (1) cell size distribution curves that are difficult to be compared numerically among large-scale samples; (2) the average size/proportion of aggregates in a population that do not specify the aggregation patterns. Here we introduce a novel application of Gini coefficient, herein named Aggregation Coefficient (AC), to quantify the aggregation levels of cystic fibrosis Pseudomonas aeruginosa (CF-PA) isolates in vitro using 3D micrographs, Fiji and MATLAB. Different aggregation patterns of five strains were compared statistically using the numerical AC indexes, which correlated well with the size distribution curves plotted by different biovolumes of aggregates. To test the sensitivity of AC, aggregates of the same strains were treated with nitric oxide (NO), a dispersal agent that reduces the biomass of surface attached biofilms. Strains unresponsive to NO were reflected by comparable AC indexes, while those undergoing dispersal showed a significant reduction in AC index, mirroring the changes in average aggregate sizes and proportions. Therefore, AC provides simpler and more descriptive numerical outputs for measuring different aggregation patterns compared to current approaches.

15 different antibiotics were individually mixed with commercially available calcium sulfate bone void filler beads. The antibiotics were: amikacin, ceftriaxone, cefuroxime, ciprofloxacin, clindamycin, colistamethate sodium, daptomycin, gentamicin, imipenem/cilastatin, meropenem, nafcillin, rifampicin, teicoplanin, tobramycin and vancomycin. The efficacy of specific released antibiotics was validated by zone of inhibition (ZOI) testing using a modified Kirby⁻Bauer disk diffusion method against common periprosthetic joint infection pathogens. With a subset of experiments (daptomycin, rifampin, vancomycin alone and rifampin and vancomycin in combination), we investigated how release varied over 15 days using a repeated ZOI assay. We also tested the ability of these beads to kill biofilms formed by Staphylococcus epidermidis 35984, a prolific biofilm former. The results suggested that certain antibiotics could be combined and released from calcium sulfate with retained antibacterial efficacy. The daptomycin and rifampin plus vancomycin beads showed antimicrobial efficacy for the full 15 days of testing and vancomycin in combination with rifampin prevented resistant mutants. In the biofilm killing assay, all of the antibiotic combinations showed a significant reduction in biofilm bacteria after 24 h. The exposure time was an important factor in the amount of killing, and varied among the antibiotics.

Aim Bacterial biofilms play a key role in prosthetic infection (PI) pathogenesis. Establishment of the biofilm phenotype confers the bacteria with significant tolerance to systemic antibiotics and ...

Biofilms are major contributors to disease chronicity and are typically multi-species in nature. Pseudomonas aeruginosa and Staphylococcus aureus are leading causes of morbidity and mortality in a variety of chronic diseases but current in vitro dual-species biofilms models involving these pathogens are limited by short co-culture times (24 to 48 hours). Here, we describe the establishment of a stable (240 hour) co-culture biofilm model of P. aeruginosa and S. aureus that is reproducible and more representative of chronic disease. The ability of two P. aeruginosa strains, (PAO1 and a cystic fibrosis isolate, PA21), to form co-culture biofilms with S. aureus was investigated. Co-culture was stable for longer periods using P. aeruginosa PA21 and S. aureus viability within the model improved in the presence of exogenous hemin. Biofilm co-culture was associated with increased tolerance of P. aeruginosa to tobramycin and increased susceptibility of S. aureus to tobramycin and a novel antimicrobial, HT61, previously shown to be more effective against non-dividing cultures of Staphylococcal spp. Biofilm growth was also associated with increased short-term mutation rates; 10-fold for P. aeruginosa and 500-fold for S. aureus. By describing a reproducible 240 hour co-culture biofilm model of P. aeruginosa and S. aureus, we have shown that interspecies interactions between these organisms may influence short-term mutation rates and evolution, which could be of importance in understanding the adaptive processes that lead to the development of antimicrobial resistance.

Biofilm formation is a key virulence factor for a wide range of microorganisms that cause chronic infections. The multifactorial nature of biofilm development and drug tolerance imposes great challenges for the use of conventional antimicrobials and indicates the need for multi-targeted or combinatorial therapies. In this Review, we focus on current therapeutic strategies and those under development that target vital structural and functional traits of microbial biofilms and drug tolerance mechanisms, including the extracellular matrix and dormant cells. We emphasize strategies that are supported by in vivo or ex vivo studies, highlight emerging biofilm-targeting technologies and provide a rationale for multi-targeted therapies aimed at disrupting the complex biofilm microenvironment.

Background: The role of Propionibacterium acnes in shoulder arthroplasty and broadly in orthopedic prosthetic infections has historically been underestimated, with biofilm formation identified as a key virulence factor attributed to invasive isolates. With an often indolent clinical course, P acnes infection can be difficult to detect and treat. This study investigates absorbable cements loaded with a broad-spectrum antibiotic combination as an effective preventive strategy to combat P acnes biofilms.Methods: P acnes biofilm formation on an unloaded synthetic calcium sulfate (CaSO4) bone void filler cement bead was evaluated by scanning electron microscopy over a period of 14 days. Beads loaded with tobramycin alone or vancomycin alone (as comparative controls) and beads loaded with a vancomycin-tobramycin dual treatment were assessed for their ability to eradicate planktonic P acnes, prevent biofilm formation, and eradicate preformed biofilms using a combination of viable-cell counts, confocal microscopy, and scanning electron microscopy.Results: P acnes surface colonization and biofilm formation on unloaded CaSO4 beads was slow. Beads loaded with antibiotics were able to kill planktonic cultures of 106 colony-forming units/mL, prevent bacterial colonization, and significantly reduce biofilm formation over periods of weeks. Complete eradication of established biofilms was achieved with a contact time of 1 week.Conclusions: This study demonstrates that antibiotic-loaded CaSO4 beads may represent an effective antibacterial and antibiofilm strategy to combat prosthetic infections in which P acnes is involved. (C) 2017 Journal of Shoulder and Elbow Surgery Board of Trustees. All rights reserved.

cystic fibrosis (CF) airway but limited evidence for this in non-CF bronchiectasis (Bx).We compared in vitro biofilm-forming behaviour of Pa strains from Bx and CF patients and explored potential confounding by bacterial growth rates.Methods: Pa strains from airway samples of 40 CF and 39 Bx patients with chronic infection were inoculated at an optical density (OD 600 ) of 0.05 into 4 replicate wells of a 96-well plate and a parallel culture tube and statically incubated for 6 hours.Adherent biomass was stained with crystal violet (CV) in the 96-well plates, eluted by ethanol and absorbance at 550 nm (A550) measured; biofilm formation was defined as staining >3SD above broth only wells.Parallel cultures were vortexed and change in OD 600 recorded as a surrogate for growth.Results: Differences in the proportions of strains demonstrating biofilm formation did not reach statistical significance between the two diseases (CF 75%; Bx 62%, Χ 2 p = 0.2).CF strains had a mean 1.7-fold greater CV staining than Bx strains ( p = 0.03).Change in OD 600 correlated with the CV biomass assay (Spearman r 0.71, 95% CI 0.57-0.81,p < 0.0001) confirming that bacterial growth contributes to this signal, but increased CV staining in CF strains was present after correcting for growth ( p = 0.04).Conclusion: Pa from CF airway cultures show increased biomass in a conventional biofilm assay when compared to similar strains from Bx patients.This difference may reflect phenotypic adaptation to an altered environment within the CF lung.Care should be taken to consider bacterial growth as an important factor in biomass formation as it may confound data generated from this assay.

Antibiotic loaded cement beads are commonly used for the treatment of biofilm related orthopaedic periprosthetic infections; however the effects of antibiotic loading and exposure of beads to body fluids on release kinetics are unclear. The purpose of this study was to determine the effects of (i) antibiotic loading density (ii) loading amount (iii) material type and (iv) exposure to body fluids (blood or synovial fluid) on release kinetics and efficacy of antibiotics against planktonic and lawn biofilm bacteria. Short-term release into an agar gel was evaluated using a fluorescent tracer (fluorescein) incorporated in the carrier materials calcium sulfate (CaSO4) and poly methyl methacrylate (PMMA). Different fluorescein concentrations in CaSO4 beads were evaluated. Mechanical properties of fluorescein-incorporated beads were analyzed. Efficacy of the antibiotics vancomycin (VAN) or tobramycin (TOB) alone and in combination was evaluated against lawn biofilms of bioluminescent strains of Staphylococcus aureus and Pseudomonas aeruginosa. Zones of inhibition of cultures (ZOI) were measured visually and using an in-vivo imaging system (IVIS). The influence of body fluids on release was assessed using CaSO4 beads that contained fluorescein or antibiotics and were pre-coated with human blood or synovial fluid. The spread from the beads followed a square root of time relationship in all cases. The loading concentration had no influence on short-term fluorescein release and pre-coating of beads with body fluids did not affect short-term release or antibacterial activity. Compared to PMMA, CaSO4 had a more rapid short term rate of elution and activity against planktonic and lawn biofilms. This study highlights the importance of considering antibiotic loading and packing density when investigating the clinical application of bone cements for infection management.

Non-typeable Haemophilus influenzae (NTHi) is an opportunistic pathogen that plays a major role in a number of respiratory tract infections, including otitis media, cystic fibrosis and chronic obstructive pulmonary disease. Biofilm formation has been implicated in both NTHi colonization and disease, and is responsible for the increased tolerance of this pathogen towards antibiotic treatment. Targeting metabolic pathways that are important in NTHi biofilm formation represents a potential strategy to combat this antibiotic recalcitrance. A previous investigation demonstrated increased expression of a putative d-methionine uptake protein following exposure of NTHi biofilms to the ubiquitous signalling molecule, nitric oxide. We therefore hypothesized that treatment with exogenous d-methionine would impact on NTHi biofilm formation and increase antibiotic sensitivity. Treatment of NTHi during the process of biofilm formation resulted in a reduction in biofilm viability, increased biomass, changes in the overall biofilm architecture and the adoption of an amorphous cellular morphology. Quantitative proteomic analyses identified 124 proteins that were differentially expressed following d-methionine treatment, of which 51 (41 %) were involved in metabolic and transport processes. Nine proteins involved in peptidoglycan synthesis and cell division showed significantly increased expression. Furthermore, d-methionine treatment augmented the efficacy of azithromycin treatment and highlighted the potential of d-methionine as an adjunctive therapeutic approach for NTHi biofilm-associated infections.

Despite aggressive antibiotic therapy, bronchopulmonary colonization by Pseudomonas aeruginosa causes persistent morbidity and mortality in cystic fibrosis (CF). Chronic P. aeruginosa infection in the CF lung is associated with structured, antibiotic-tolerant bacterial aggregates known as biofilms. We have demonstrated the effects of non-bactericidal, low-dose nitric oxide (NO), a signaling molecule that induces biofilm dispersal, as a novel adjunctive therapy for P. aeruginosa bioffim infection in CF in an ex vivo model and a proof-of-concept double-blind clinical trial. Submicromolar NO concentrations alone caused disruption of biofilms within ex vivo CF sputum and a statistically significant decrease in ex vivo biofilm tolerance to tobramycin and tobramycin combined with ceftazidime. In the 12-patient randomized clinical trial, 10 ppm NO inhalation caused significant reduction in P. aeruginosa biofilm aggregates compared with placebo across 7 days of treatment. Our results suggest a benefit of using low dose NO as adjunctive therapy to enhance the efficacy of antibiotics used to treat acute P. aeruginosa exacerbations in CF. Strategies to induce the disruption of bioffims have the potential to overcome biofilm-associated antibiotic tolerance in CF and other biofilm-related diseases.

Biofilms are thin layers of bacteria embedded within a slime matrix that live on surfaces. They are ubiquitous in nature and responsible for many medical and dental infections, industrial fouling and are also evident in ancient fossils. A biofilm structure is shaped by growth, detachment and response to mechanical forces acting on them. The main contribution to biofilm versatility in response to physical forces is the matrix that provides a platform for the bacteria to grow. The interaction between biofilm structure and hydrodynamics remains a fundamental question concerning biofilm dynamics. Here, we document the appearance of ripples and wrinkles in biofilms grown from three species of bacteria when subjected to high-velocity fluid flows. Linear stability analysis suggested that the ripples were Kelvin-Helmholtz Instabilities. The analysis also predicted a strong dependence of the instability formation on biofilm viscosity explaining the different surface corrugations observed. Turbulence through Kelvin-Helmholtz instabilities occurring at the interface demonstrated that the biofilm flows like a viscous liquid under high flow velocities applied within milliseconds. Biofilm fluid-like behavior may have important implications for our understanding of how fluid flow influences biofilm biology since turbulence will likely disrupt metabolite and signal gradients as well as community stratification.

Biofilm formation represents a key stage in the pathogenesis of prosthetic infections (PIs). More tolerant to antibiotics than their planktonic counterparts, biofilm bacteria are difficult to eradicate using conventional therapeutic regimes. A common approach in PI management is the adjunctive use of localised antibiotics in addition to systemic administration in an attempt to protect the implant from colonisation by infiltrating bacteria. This study evaluates the antibacterial and antibiofilm efficacy of antibiotic-loaded dissolvable calcium sulphate, previously shown to be effective against key gram-positive pathogens, against gram-negative species important in the establishment of chronic infection in PIs. Synthetic calcium sulfate beads loaded with tobramycin, vancomycin and both antibiotics in combination were assessed for their ability to eradicate planktonic Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae strains. The efficacy of the beads in preventing biofilm formation and eliminating established biofilms over multiple days was evaluated using confocal laser scanning microscopy (CSLM) imaging combined with image analysis and viable cell counts. Beads loaded with antibiotics demonstrated effective eluting concentrations for up to 37 d depending on the bacterial strain. In the presence of repeated bacterial challenges, antibiotic-loaded beads prevented bacterial colonisation and significantly reduce biofilm formation for the duration of the assay (7 d). Complete eradication of established biofilms was more difficult with evidence of biofilm regrowth after 1 week of contact with antibiotic-loaded beads, despite data suggesting a complete kill was achieved at earlier timepoints of 24 h and 72 h in the case of K. pneumoniae and P. aeruginosa. This study provides further evidence that calcium sulfate beads loaded with vancomycin and tobramycin may be a useful adjunctive component to the successful management of PIs.

Streptococcus pneumoniae is a commensal human pathogen and the causative agent of various invasive and noninvasive diseases. Carriage of the pneumococcus in the nasopharynx is thought to be mediated by biofilm formation, an environment where isogenic populations frequently give rise to morphological colony variants, including small colony variant (SCV) phenotypes. We employed metabolic characterization and whole-genome sequencing of biofilm-derived S. pneumoniae serotype 22F pneumococcal SCVs to investigate diversification during biofilm formation. Phenotypic profiling revealed that SCVs exhibit reduced growth rates, reduced capsule expression, altered metabolic profiles, and increased biofilm formation compared to the ancestral strain. Whole-genome sequencing of 12 SCVs from independent biofilm experiments revealed that all SCVs studied had mutations within the DNA-directed RNA polymerase delta subunit (RpoE). Mutations included four large-scale deletions ranging from 51 to 264 bp, one insertion resulting in a coding frameshift, and seven nonsense single-nucleotide substitutions that result in a truncated gene product. This work links mutations in the rpoE gene to SCV formation and enhanced biofilm development in S. pneumoniae and therefore may have important implications for colonization, carriage, and persistence of the organism. Furthermore, recurrent mutation of the pneumococcal rpoE gene presents an unprecedented level of parallel evolution in pneumococcal biofilm development.

Bacterial infections of central venous catheters (CVCs) cause much morbidity and mortality, and are usually diagnosed by concordant culture of blood and catheter tip. However, studies suggest that culture often fails to detect biofilm bacteria. This study optimizes X-ray micro-focus computed tomography (X-ray µCT) for the quantification and determination of distribution and heterogeneity of biofilms in in vitro CVC model systems. Bacterial culture and scanning electron microscopy (SEM) were used to detect Staphylococcus epidermidis ATCC 35984 biofilms grown on catheters in vitro in both flow and static biofilm models. Alongside this, X-ray µCT techniques were developed in order to detect biofilms inside CVCs. Various contrast agent stains were evaluated using energy-dispersive X-ray spectroscopy (EDS) to further optimize these methods. Catheter material and biofilm were segmented using a semi-automated matlab script and quantified using the Avizo Fire software package. X-ray µCT was capable of distinguishing between the degree of biofilm formation across different segments of a CVC flow model. EDS screening of single- and dual-compound contrast stains identified 10 nm gold and silver nitrate as the optimum contrast agent for X-ray µCT. This optimized method was then demonstrated to be capable of quantifying biofilms in an in vitro static biofilm formation model, with a strong correlation between biofilm detection via SEM and culture. X-ray µCT has good potential as a direct, non-invasive, non-destructive technology to image biofilms in CVCs, as well as other in vivo medical components in which biofilms accumulate in concealed areas.

Streptococcus pneumoniae is a commensal human pathogen and the causative agent of various invasive and noninvasive diseases. Carriage of the pneumococcus in the nasopharynx is thought to be mediated by biofilm formation, an environment where isogenic populations frequently give rise to morphological colony variants, including small colony variant (SCV) phenotypes. We employed metabolic characterization and whole-genome sequencing of biofilm-derived S. pneumoniae serotype 22F pneumococcal SCVs to investigate diversification during biofilm formation. Phenotypic profiling revealed that SCVs exhibit reduced growth rates, reduced capsule expression, altered metabolic profiles, and increased biofilm formation compared to the ancestral strain. Whole-genome sequencing of 12 SCVs from independent biofilm experiments revealed that all SCVs studied had mutations within the DNA-directed RNA polymerase delta subunit (RpoE). Mutations included four large-scale deletions ranging from 51 to 264 bp, one insertion resulting in a coding frameshift, and seven nonsense single-nucleotide substitutions that result in a truncated gene product. This work links mutations in the rpoE gene to SCV formation and enhanced biofilm development in S. pneumoniae and therefore may have important implications for colonization, carriage, and persistence of the organism. Furthermore, recurrent mutation of the pneumococcal rpoE gene presents an unprecedented level of parallel evolution in pneumococcal biofilm development.

Chronic rhinosinusitis (CRS) with or without nasal polyps (NPs) (CRSwNP and CRSsNP, respectively) is one of the most common conditions encountered in medicine.1Fokkens W.J. Lund V.J. Mullol J. Bachert C. Alobid I. Baroody F. et al.European position paper on rhinosinusitis and nasal polyps 2012.Rhinology. 2012; 23: 1-298Google Scholar CRS is a disease of the mucosa lining the sinonasal cavity characterized by recurrent episodes of inflammation resulting in chronic symptoms such as nasal obstruction, facial pain, rhinorrhea, and reduction in sense of smell.1Fokkens W.J. Lund V.J. Mullol J. Bachert C. Alobid I. Baroody F. et al.European position paper on rhinosinusitis and nasal polyps 2012.Rhinology. 2012; 23: 1-298Google Scholar CRS affects up to 15% of the general population in Europe and the United States, ranking it second in prevalence among chronic conditions,1Fokkens W.J. Lund V.J. Mullol J. Bachert C. Alobid I. Baroody F. et al.European position paper on rhinosinusitis and nasal polyps 2012.Rhinology. 2012; 23: 1-298Google Scholar and significantly affecting quality of life and health care resources.2Cohen M. Kofonow J. Nayak J.V. Palmer J.N. Chiu A.G. Leid J.G. et al.Biofilms in chronic rhinosinusitis: a review.Am J Rhinol Allergy. 2009; 23: 255-260Crossref PubMed Scopus (85) Google Scholar Despite the massive expenditure on medical and surgical therapies for this condition, a subset of patients remains resistant to all established treatments.2Cohen M. Kofonow J. Nayak J.V. Palmer J.N. Chiu A.G. Leid J.G. et al.Biofilms in chronic rhinosinusitis: a review.Am J Rhinol Allergy. 2009; 23: 255-260Crossref PubMed Scopus (85) Google Scholar Identification of either a host or an environmental cause has been unsuccessful. Proposed mechanisms of CRS pathophysiology include the role of superantigens, abnormal cell-mediated immune responses, changes in the inflammatory cytokine cascade, epithelial defects, osteitis of the sinus walls, and viral, bacterial, and fungal factors.1Fokkens W.J. Lund V.J. Mullol J. Bachert C. Alobid I. Baroody F. et al.European position paper on rhinosinusitis and nasal polyps 2012.Rhinology. 2012; 23: 1-298Google Scholar Recent guidelines propose the classification of CRSwNP and CRSsNP into 2 distinct pathological entities primarily based on differences in inflammatory cytokine profiles.1Fokkens W.J. Lund V.J. Mullol J. Bachert C. Alobid I. Baroody F. et al.European position paper on rhinosinusitis and nasal polyps 2012.Rhinology. 2012; 23: 1-298Google Scholar A CRSwNP TH2-mediated profile is characterized as eosinophilic with elevated IL-5, IgE, RANTES, and eotaxin,1Fokkens W.J. Lund V.J. Mullol J. Bachert C. Alobid I. Baroody F. et al.European position paper on rhinosinusitis and nasal polyps 2012.Rhinology. 2012; 23: 1-298Google Scholar and a CRSsNP TH1-mediated profile is characterized as neutrophilic with elevated TNF-α, IL-8, and IFN-γ.1Fokkens W.J. Lund V.J. Mullol J. Bachert C. Alobid I. Baroody F. et al.European position paper on rhinosinusitis and nasal polyps 2012.Rhinology. 2012; 23: 1-298Google Scholar Emerging evidence implicates bacterial biofilms as mediators of the inflammatory reaction in CRS.1Fokkens W.J. Lund V.J. Mullol J. Bachert C. Alobid I. Baroody F. et al.European position paper on rhinosinusitis and nasal polyps 2012.Rhinology. 2012; 23: 1-298Google Scholar Several studies provide strong evidence that bacterial biofilms perpetuate inflammation in CRS and are associated with more severe preoperative disease, persistence of ongoing mucosal inflammation, and poor postsurgical outcomes.3Singhal D. Psaltis A.J. Foreman A. Wormald P.J. The impact of biofilms on outcomes after endoscopic sinus surgery.Am J Rhinol Allergy. 2010; 24: 169-174Crossref PubMed Scopus (106) Google Scholar Staphylococcus aureus has been identified as the commonest biofilm-forming microbe in CRS and colonizes the sinonasal cavities in 27% of the patients with CRSsNP and in 60% of the patients with CRSwNP.4Bachert C. van Zele T. Gevaert P. de Schrijver L. Van Cauwenberge P. Superantigens and nasal polyps.Curr Allergy Asthma Rep. 2003; 3: 523-531Crossref PubMed Scopus (90) Google Scholar Around 20% of the patients with CRS develop NPs,1Fokkens W.J. Lund V.J. Mullol J. Bachert C. Alobid I. Baroody F. et al.European position paper on rhinosinusitis and nasal polyps 2012.Rhinology. 2012; 23: 1-298Google Scholar and although studies have clearly identified bacterial biofilms on the sinonasal mucosa of patients with CRS, little data pertain to the bacterial profiles specifically in NPs. We conducted a preliminary study characterizing bacterial profiles in NPs, comparing them with those on nonpolypoidal sinonasal mucosa from the same patients and with non-CRS sinonasal mucosa as control tissue. A prospective study with full ethical approval was conducted in 9 patients with CRSwNP undergoing functional endoscopic sinus surgery and 5 control patients undergoing transsphenoidal pituitary surgery. Nonpolypoidal sinonasal mucosa and NPs were collected from each patient with CRSwNP and sinonasal mucosa from controls. The bacterial profiles were assessed using fluorescence in situ hybridization with confocal laser scanning microscopy (CLSM) and immunohistochemistry. Hybridization conditions were optimized for CRS tissue using appropriate controls (see the Methods section in this article's Online Repository at www.jacionline.org). CLSM demonstrated surface-related bacterial biofilms on the nonpolypoidal sinonasal mucosa of all 9 CRS samples (Fig 1, A), but not on the epithelial surface of NPs. However, subepithelial and intracellular bacteria were observed in the cytoplasm of host cells in all 9 NP samples (Fig 1, B). The CLSM Z-axis view indicated that intracellular bacteria were subepithelial in all cases (Fig 1, C). Bacteria were confirmed within host cells using 4′,6-diamidino-2-phenylindole, a nucleic acid stain that resolves the host cell nucleus, surrounded by densely packed bacteria filling the cytoplasm (Fig 1, D). Biofilms were not observed in any control samples. Species-specific fluorescence in situ hybridization identified brightly fluorescent bacteria in CRS samples including S aureus in 78%, Haemophilus influenza in 33%, and Pseudomonas aeruginosa in 33% of the patients (see Table E1 in this article's Online Repository at www.jacionline.org). Immunohistochemistry confirmed our finding of subepithelial intracellular S aureus, using an S aureus mAb (Fig 1, E). Immunohistochemical colocalization on sequential sections identified the S aureus–harboring host cells as mast cells (MCs) (Fig 2, A-D).Fig 2Immunohistochemical colocalization. Photomicrographs of sequential 2-μm sections of NP tissue stained with anti-AA1 MC tryptase (A) and mouse anti–S aureus (B) demonstrating subepithelial intracellular S aureus within MCs (arrows) (×20 magnification). Both images are shown at higher magnification (C and D ×40 magnification).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Our study directly compared nonpolypoidal sinonasal mucosa with NPs from the same patient (thus controlling for host genetics) and is the first to observe both subepithelial bacteria in ex vivo NP tissue and intracellular localization of S aureus within MCs. This contrasts with recent studies that have shown intracellular S aureus in nonpolypoidal CRS sinonasal mucosa.5Tan N.C. Foreman A. Jardeleza C. Douglas R. Tran H. Wormald P.J. The multiplicity of Staphylococcus aureus in chronic rhinosinusitis: correlating surface biofilm and intracellular residence.Laryngoscope. 2012; 122: 1655-1660Crossref PubMed Scopus (52) Google Scholar Sachse et al6Sachse F. Becker K. von Eiff C. Metze D. Rudack C. Staphylococcus aureus invades the epithelium in nasal polyposis and induces IL-6 in nasal epithelial cells in vitro.Allergy. 2010; 65: 1430-1437Crossref PubMed Scopus (105) Google Scholar demonstrated intracellular S aureus replication in vitro in NP epithelial cells by introducing an S aureus strain to a cell culture model. The mechanism of bacterial migration from surface-related biofilms to intracellular reservoirs remains unclear. However, growing evidence suggests that alterations in the epithelial layer compromise its function as a defensive barrier against environmental allergens and pathogens, as well as a mediator and regulator of innate and adaptive immunity.7Soyka M.B. Wawrzyniak P. Eiwegger T. Holzmann D. Treis A. Wanke K. et al.Defective epithelial barrier in chronic rhinosinusitis: the regulation of tight junctions by IFN- and IL-4.J Allergy Clin Immunol. 2012; 130: 1087-1096Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar A reduction in tight junction proteins and increased proinflammatory cytokines IFN-γ and IL-4 within NPs lead to a disruption in epithelial integrity, induction of epithelial remodeling, and the formation of surface defects.7Soyka M.B. Wawrzyniak P. Eiwegger T. Holzmann D. Treis A. Wanke K. et al.Defective epithelial barrier in chronic rhinosinusitis: the regulation of tight junctions by IFN- and IL-4.J Allergy Clin Immunol. 2012; 130: 1087-1096Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar A defective epithelial layer may provide an opportunity for surface-related bacterial biofilms, already present in CRS, to disperse and penetrate into the tissue. This may explain the absence of surface-related biofilms observed on NP tissue in our study compared with those seen on sinonasal mucosa. Initially, the bacteria-harboring host cells were presumed to be mononuclear phagocytes, but our immunohistochemical findings rather unexpectedly confirmed them to be MCs. This constitutes a novel finding in NPs. MCs appear to play an important role in promoting innate immunity against microbial pathogens.8Stelekati E. Bahri R. D'Orlando O. Orinska Z. Mittrucker H.W. Langenhaun R. et al.Mast cell-mediated antigen presentation regulates CD8+ T cell effector functions.Immunity. 2009; 31: 665-676Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar Through activation of CD8+ T cells, MCs possess the ability to regulate both the TH1 and TH2 cytokine pathways, and can therefore adopt both immunosuppressive and immune-stimulatory properties.8Stelekati E. Bahri R. D'Orlando O. Orinska Z. Mittrucker H.W. Langenhaun R. et al.Mast cell-mediated antigen presentation regulates CD8+ T cell effector functions.Immunity. 2009; 31: 665-676Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar Recently, MCs have been demonstrated to exert phagocytosis-independent antimicrobial activity against S aureus, mediated through extracellular traps and the release of antibacterial enzymes.9Abel J. Goldmann O. Ziegler C. Holtje C. Smeltzer M.S. Cheung A.L. et al.Staphylococcus aureus evades the extracellular antimicrobial activity of mast cells by promoting its own uptake.J Innate Immun. 2011; 3: 495-507Crossref PubMed Scopus (63) Google Scholar S aureus has been shown in vitro to subvert these extracellular antimicrobial mechanisms by internalizing within MCs.9Abel J. Goldmann O. Ziegler C. Holtje C. Smeltzer M.S. Cheung A.L. et al.Staphylococcus aureus evades the extracellular antimicrobial activity of mast cells by promoting its own uptake.J Innate Immun. 2011; 3: 495-507Crossref PubMed Scopus (63) Google Scholar Once within the MCs, S aureus appears to access the nutrient-rich cytosol and upregulate cell wall synthesis, allowing persistent and viable intracellular SA reservoirs to be established.9Abel J. Goldmann O. Ziegler C. Holtje C. Smeltzer M.S. Cheung A.L. et al.Staphylococcus aureus evades the extracellular antimicrobial activity of mast cells by promoting its own uptake.J Innate Immun. 2011; 3: 495-507Crossref PubMed Scopus (63) Google Scholar The MC in the context of chronic S aureus infection may well act as a double-edged sword.9Abel J. Goldmann O. Ziegler C. Holtje C. Smeltzer M.S. Cheung A.L. et al.Staphylococcus aureus evades the extracellular antimicrobial activity of mast cells by promoting its own uptake.J Innate Immun. 2011; 3: 495-507Crossref PubMed Scopus (63) Google Scholar Although promoting innate immunity against microbial pathogens, the MC may be providing a safe haven for S aureus by protecting it from extracellular antimicrobial compounds. This not only avoids clearance but also facilitates the establishment of an intracellular microbial reservoir that could lead to persistence and chronic carriage. This may explain the high levels of resistance to systemic antibacterial therapies in chronic conditions such as CRS. Crucially, these intracellular S aureus reservoirs may constitute potential future therapeutic targets for the development of novel bacterial eradication strategies, aimed at reducing systemic antimicrobial usage, and in turn, the associated risk of antimicrobial drug resistance. To clarify mechanisms underlying survival of S aureus, mechanistic studies using in vitro MC culture models are currently being planned. We are indebted to all the study participants. We are grateful for the assistance received from staff of the Histochemistry Research and Biomedical Imaging Units. We also thank Nijaguna Mathad (Consultant Neurosurgeon, University Hospital Southampton NHS Foundation Trust [UHSNFT]) and Ashok Rokade (Consultant Rhinologist and Anterior Skull Base Surgeon, UHSNFT) for providing us with the control tissue. Nine consecutive patients with CRS with NPs, meeting the diagnostic criteria defined in the latest European position paper on rhinosinusitis and nasal polyps 2012,E1Fokkens W.J. Lund V.J. Mullol J. Bachert C. Alobid I. Baroody F. et al.European position paper on rhinosinusitis and nasal polyps 2012.Rhinology. 2012; 23: 1-298Google Scholar were included in the study. These patients underwent functional endoscopic sinus surgery by the senior authors (P.G.H. and R.J.S.) at University Hospital Southampton NHS Foundation Trust after having failed an 8-week trial of maximal medical therapy. None of the 9 patients had used either topical or systemic corticosteroid, antibacterial, or antihistamine therapies in the 8 weeks before surgery. Exclusion criteria included age less than 18 years and patients suffering from cystic fibrosis or primary ciliary dyskinesia. All subjects were skin-prick allergy tested to a standard battery of 10 common aero-allergens (ALK-Abelló, Reading, Berkshire, United Kingdom) before surgery. Radiological severity of sinus disease was graded on a computed tomography sinuses scan using the Lund-Mackay scoring system.E2Lund V.J. Mackay I.S. Staging in rhinosinusitis.Rhinology. 1993; 107: 183-184Google Scholar Preoperative demographical data were collected including age, sex, atopic status, previous sinonasal surgery, medical history, history of asthma, history of aspirin sensitivity, and smoking habits (Table E2). Control tissue was obtained from 5 patients with no clinical history or radiological evidence of CRS undergoing endoscopic trans-sphenoidal pituitary surgery at the Wessex Neurological Centre, University Hospital Southampton NHS Foundation Trust. Ethical approval was obtained from the United Kingdom NHS Research Ethics Committee (REC 09/H0501/74). All participants within the study provided informed written consent before inclusion in the study. Operative mucosal specimens and NP tissue were obtained from each patient with CRS. Matching mucosal samples were obtained from each control. A swab was taken from the middle meatus and submitted for microbiological analysis. Tissue specimens were placed in HBSS (Gibco, Paisley, United Kingdom) and transported on ice for orientation. Each sample was gently rinsed in PBS twice for 5 minutes to remove any planktonic bacteria before fixing or analysis. This was performed because planktonic bacteria (including normal nasal floral microbes) were identified on the surface of CRS and control tissue in unrinsed samples when tested with BacLight LIVE/DEAD viability nucleic acid probes. This is consistent with findings in the literature.E3Kluytmans J. van Belkum A. Verbrugh H. Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms, and associated risks.Clin Microbiol Rev. 1997; 10: 505-520Crossref PubMed Google Scholar In our study samples, gentle rinsing of the tissue twice before analysis appeared to remove planktonic bacteria but did not disrupt the bacterial biofilms. The protocol was adopted from Nistico et al,E4Nistico L. Kreft R. Gieseke A. Coticchia J.M. Burrows A. Khampang P. et al.Adenoid reservoir for pathogenic biofilm bacteria.J Clin Microbiol. 2011; 49: 1411-1420Crossref PubMed Scopus (121) Google Scholar optimized for CRS tissue. Briefly, specimens were placed in 4% paraformaldehyde in PBS followed by serial washes with PBS and finally stored at −20°C in PBS-ethanol (1:1). The 16S rRNA fluorescence in situ hybridization (FISH) probes used were as follows: Staphylococcus aureus (Sau) 5′-GAAGCAAGCTTCTCGTCCG-3′, Staphylococcus genus (Sta) 5′-TCCTCCATATCTCTGCGC-3′, Haemophilus influenza (Hinf) 5′-CCGCACTTTCATCTCCG-3′, Pseudomonas aeruginosa (PaerA) 5′-GGTAACCGTCCCCCTTGC-3′, and the universal eubacterial consensus sequence probe (Eub338) 5′-GCTGCCTCCCGTAGGAGT-3′. A maximum of 3 probes were used on each specimen to limit cross-interference with the 3 labeled fluorescent dyes, Cy3, Cy5, and 6-FAM. Because of limitations of available clinical samples and to facilitate the detection of both Gram-positive and Gram-negative bacteria simultaneously using FISH, we used a single fixation process.E4Nistico L. Kreft R. Gieseke A. Coticchia J.M. Burrows A. Khampang P. et al.Adenoid reservoir for pathogenic biofilm bacteria.J Clin Microbiol. 2011; 49: 1411-1420Crossref PubMed Scopus (121) Google Scholar, E5Nistico L. Gieseke A. Stoodley P. Hall-Stoodley L. Kerschner J.E. Ehrlich G.D. Fluorescence "in situ" hybridization for the detection of biofilm in the middle ear and upper respiratory tract mucosa.Methods Mol Biol. 2009; 493: 191-213Crossref PubMed Scopus (42) Google Scholar, E6Hall-Stoodley L. Hu F.Z. Gieseke A. Nistico L. Nguyen D. Hayes J. et al.Direct detection of bacterial biofilms on the middle-ear mucosa of children with chronic otitis media.JAMA. 2006; 296: 202-211Crossref PubMed Scopus (693) Google Scholar Samples were incubated with 0.5 mg/mL of lysozyme (Sigma-Aldrich, St Louis, Mo) followed by incubation with the probes mixed with hybridization buffer, containing 1 mol Tris HCl, 5 mol NaCl, 20% formamide, and 10% SDS, for 2 hours at 48°C. Finally, samples were submerged in wash buffer at 46°C for 15 minutes, followed by immersion in PBS and visualized using CLSM. In this study, the hybridization conditions including formamide concentration for all the 16S rRNA probes were optimized using common respiratory bacterial isolates and using tissue samples as described in previous publications.E4Nistico L. Kreft R. Gieseke A. Coticchia J.M. Burrows A. Khampang P. et al.Adenoid reservoir for pathogenic biofilm bacteria.J Clin Microbiol. 2011; 49: 1411-1420Crossref PubMed Scopus (121) Google Scholar, E5Nistico L. Gieseke A. Stoodley P. Hall-Stoodley L. Kerschner J.E. Ehrlich G.D. Fluorescence "in situ" hybridization for the detection of biofilm in the middle ear and upper respiratory tract mucosa.Methods Mol Biol. 2009; 493: 191-213Crossref PubMed Scopus (42) Google Scholar, E6Hall-Stoodley L. Hu F.Z. Gieseke A. Nistico L. Nguyen D. Hayes J. et al.Direct detection of bacterial biofilms on the middle-ear mucosa of children with chronic otitis media.JAMA. 2006; 296: 202-211Crossref PubMed Scopus (693) Google Scholar Briefly, fluorescently conjugated 16S rRNA probes were applied to fixed, permeabilized bacterial cells under stringent hybridization conditions for each probe. Stringency conditions were achieved by changing the formamide concentration incrementally by 5% from 0% to 50% together with the salt concentrations, while keeping hybridization and wash temperatures constant. Optimal stringency was empirically determined to be the highest formamide concentration not resulting in loss of fluorescent intensity on the target pure culture bacterial representative species. Nonspecific hybridization of oligonucleotide probes was not observed with any clinical isolates of Streptococcus pneumoniae, H influenza, Moraxella catarrhalis, P aeruginosa, and S aureus tested. All isolates hybridized with the eubacterial 16S rRNA probe (Table E2). CLSM imaging was performed as previously described.E5Nistico L. Gieseke A. Stoodley P. Hall-Stoodley L. Kerschner J.E. Ehrlich G.D. Fluorescence "in situ" hybridization for the detection of biofilm in the middle ear and upper respiratory tract mucosa.Methods Mol Biol. 2009; 493: 191-213Crossref PubMed Scopus (42) Google Scholar Briefly, after staining, tissues were mounted (IBIDI) within chamber slides with cover-slip bottoms and imaged with a Leica TCS SP5 inverted confocal system (Leica Microsystems, Milton Keynes, United Kingdom) using a 63× oil immersion lens. Sequential scanning was used to further eliminate cross-talk interference from multiple fluorophores. Images were collected and analyzed using Leica LAS-AF software. The immunohistochemistry protocol was adopted from Britten et al.E7Britten K.M. Howarth P.H. Roche W.R. Immunohistochemistry on resin sections: a comparison of resin embedding techniques for small mucosal biopsies.Biotech Histochem. 1993; 68: 271-280Crossref PubMed Scopus (199) Google Scholar Briefly, immunohistochemistry was applied to glycol methacrylate embedded tissue. Primary antibodies were applied for 20 hours at previously titrated optimal dilutions (Table E3). Appropriate biotinylated secondary antibodies (Dako, Ely, United Kingdom) were then applied for 2 hours followed by application of avidin biotin-peroxide complexes. Previously titrated chromogen 3,3′-Diaminobenzidine substrate was then applied, and finally the sections were counterstained with Mayer haematoxylin. All experiments included negative control slides with omission of primary antibodies and appropriate isotype-matched antibodies. A positive control model using an isolated CRS S aureus strain was used to test the accuracy of the mouse anti–S aureus primary antibody (Abcam, Cambridge, United Kingdom) and to optimize the concentration of DAB substrate, before use on study sections. Colocalization was performed using sequential adjacent 2-μm tissue sections immunostained with appropriate comparable primary antibodies. For each patient and tissue type (sinonasal mucosa or nasal polyp), 5 specimens were analyzed for bacterial biofilms or intracellular bacteria. Bacteria were defined as being within a biofilm when aggregated in clusters and attached to the sinonasal mucosa.E8Parsek M.R. Singh P.K. Bacterial biofilms: an emerging link to disease pathogenesis.Annu Rev Microbiol. 2003; 57: 677-701Crossref PubMed Scopus (1202) Google Scholar The biofilm detection rate represents the number of biofilm positive specimens (α) identified from 5 tissue samples (biofilm detection rate = α/5). The intracellular bacteria detection rate represents the number of specimens with observed subepithelial intracellular bacteria (β) identified from 5 tissue samples (intracellular bacteria detection rate = β/5). Statistical analysis was performed using Graph Pad Prism 6.0 software (Graph Pad Software, Inc, San Diego, Calif). Comparisons between clinical groups for FISH, where the mean of the control groups was 0, were made by using the 1-sample t test (2 tailed). The level of significance was accepted as a P value of less than .05.Table E1Biofilm and intracellular bacterial detection rates for NP tissuePatient no.Surface biofilms (BDR)Staphylococcus aureus (IBDR)Pseudomonas aeruginosa (IBDR)Haemophilus influenzae (IBDR)Eubacterial probeCRSwNP group 10.00.60.00.0+ 20.00.60.00.0+ 30.00.00.60.2+ 40.00.80.00.0+ 50.01.00.40.2+ 60.00.40.00.0+ 70.00.00.60.6+ 80.00.40.00.0+ 90.00.60.00.0+Total07/9 (78%)3/9 (33%)3/9 (33%)Mean0.00.490.180.11P value.0.002∗P < .05..086.13995% CI0.00.23-0.34−0.03 to 0.34−0.04 to 0.27Control group 10.00.00.00.0− 20.00.00.00.0− 30.00.00.00.0− 40.00.00.00.0− 50.00.00.00.0−Mean0.00.00.00.0BDR, Biofilm detection rate; IBDR, intracellular bacterial detection rate; +, detected; −, undetected.∗ P < .05. Open table in a new tab Table E2Demographics and microbial profile of study populationPatientSex (age [y])SPTSHASAHCCBacteria detected by FISHLMSStaphylococcus aureusStaphylococcus genusHaemophilus influenzaePseudomonas aeruginosaEubacterial probeCRSwNP 1F (48)−+−−−++−−+10 2F (20)−−−−−++−−+13 3M (64)−+−−+ C−−+++9 4F (21)−−−−−++−−+15 5F (24)−+−−+ CNS+++++22 6F (56)+ G+−++ C++−−+19 7F (73)−−−−+ P aeruginosa−−+++14 8M (63)−−−+−++−−+15 9M (40)−−−−−++−−+16Controls 1F (73)−−−−−−−−−−0 2F (76)−−−−−−−−−−0 3M (52)−−−−−−−−−−0 4M (47)−−−−−−−−−−0 5M (45)−−−−−−−−−−0AH, Asthma history; AS, aspirin sensitivity; C, coliforms; CC, conventional culture; CNS, coagulase-negative Staphylococcus; F, female; G, grass; LMS, Lund-Mackay Score; M, male; SH, smoking history; SPT, skin prick testing; +, detected; −, undetected. Open table in a new tab Table E3Summary of primary and secondary antibodies, sources, species, and working dilutions with DAB as substratePrimary antibodyCloneSourceHost speciesWorking dilutionSecondary antibodyWorking dilutionMC tryptaseAA1Abcam, Cambridge, UK1:20000NeutrophilsNOEDako, Ely, UK1:1000EosinophilsEG2Life Technologies, Paisely, UK1:2000MacrophagesCD68PGMIDako, Ely, UKMouse1:100Rabbit antimouse (Dako, Ely, UK)1:1000T LymphocytesCD3AbD Serotec, Oxford, UK1:700B LymphocytesCD20Dako, Ely, UK1:2000Epithelial cellsPanCKSigma-Aldrich, St Louis, Mo1:4000DAB, 3,3′-Diaminobenzidine; UK, United Kingdom. Open table in a new tab BDR, Biofilm detection rate; IBDR, intracellular bacterial detection rate; +, detected; −, undetected. AH, Asthma history; AS, aspirin sensitivity; C, coliforms; CC, conventional culture; CNS, coagulase-negative Staphylococcus; F, female; G, grass; LMS, Lund-Mackay Score; M, male; SH, smoking history; SPT, skin prick testing; +, detected; −, undetected. DAB, 3,3′-Diaminobenzidine; UK, United Kingdom.

Introduction Chronic respiratory infection is responsible for the majority of the morbidity and mortality of CF patients. In order to guide treatment regimes and improve understanding of the pathophysiology of CF, airway secretions are sampled and analysed. Sputum is usually the selected method of sampling. Often, large quantities of sputum are required to facilitate comprehensive laboratory testing. Hence, when designing studies, it is important to consider the quantity of sputum likely to be produced by patients and to ensure that the composition of the sputum samples is not altered by the procedure by which they are obtained. This study aimed to investigate the effect of chest physiotherapy on the quantity and composition of sputum samples collected from individuals with CF. It was hypothesised that physiotherapy would increase the quantity of sputum produced, reduce the salivary content and alter the microbiological content. Methods Clinically stable adults with CF were recruited at outpatient clinics and randomised into group A (physiotherapy group, n = 21) or group B (no physiotherapy group, n = 25). Laboratory processing of the samples involved determining sample weights and counting human cells (alive respiratory cells, dead respiratory cells and squamous cells). The dissolved sputum was also transferred onto plates of cetrimide agar for culturing Pseudomonas aeruginosa. Colony-forming units (CFUs) were counted on the plates after 24 h. Results Samples from the physiotherapy group had significantly greater weights than the no physiotherapy group (p < 0.001). When considering the total number of cells per gram of sputum, there was no statistical difference between the two groups (p = 0.396). However, the numbers of squamous cells per gram, and dead respiratory cells per gram were both significantly greater in the no physiotherapy group (p = 0.039 and p = 0.001 respectively). There were no significant differences between numbers of alive respiratory cells per gram (p = 0.487) or CFUs (p = 0.459). Conclusion Whilst physiotherapy was found to increase the quantity of sputum collected, there were significant differences in sputum composition, suggesting that the two groups represent samples from different niches. Hence, when planning a study involving sputum analysis, the procedure by which the sample is obtained has to be considered when interpreting the results.

As the number of total joint arthroplasty and internal fixation procedures continues to rise, the threat of infection following surgery has significant clinical implications. These infections may have highly morbid consequences to patients, who often endure additional surgeries and lengthy exposures to systemic antibiotics, neither of which are guaranteed to resolve the infection. Of particular concern is the threat of bacterial biofilm development, since biofilm-mediated infections are difficult to diagnose and effective treatments are lacking. Developing therapeutic strategies have targeted mechanisms of biofilm formation and the means by which these bacteria communicate with each other to take on specialized roles such as persister cells within the biofilm. In addition, prevention of infection through novel coatings for prostheses and the local delivery of high concentrations of antibiotics by absorbable carriers has shown promise in laboratory and animal studies. Biofilm development, especially in an arthoplasty environment, and future diagnostic and treatment options are discussed.