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Objective The incidence of refractory chronic rhinosinusitis (CRS) associated with methicillin-resistant Staphylococcus aureus (MRSA) is rising and remains a therapeutic challenge. The goal of this study is to demonstrate the efficacy of a non-invasive topical therapy against MRSA in these patients. Methods Seventeen patients with refractory CRS caused by MRSA were treated with a topical therapy protocol. Treatment consisted of weekly endoscopic sinus debridement followed by intra-sinus installation of a hydroxyl-ethylcellulose gel that releases mometasone and a culture-directed antibiotic for a period of 6 weeks, along with daily nasal nebulization of mometasone with the same antibiotic and saline rinses. Clinical outcome was assessed using the Lund–Kennedy (LK) symptom and endoscopic appearance scores. Sinus mucosal tissue was homogenized and cultured, and microbial biofilm burden was assessed based on colony forming units (CFUs) counts. Results Rhinotopic therapy resulted in clearance of MRSA in 13 of 16 patients (81.2%). Treated patients also demonstrated significant improvement clinically as measured by the LK scores. In addition, a significant decrease in mucosal CFUs was observed post-therapy. Conclusion Our findings demonstrate that topical therapy is an effective method for treating MRSA-associated refractory CRS.

Staphylococcus aureus is a causative agent of chronic biofilm-associated infections that are recalcitrant to resolution by the immune system or antibiotics. To combat these infections, an antistaphylococcal, biofilm-specific quadrivalent vaccine against an osteomyelitis model in rabbits has previously been developed and shown to be effective at eliminating biofilm-embedded bacterial populations. However, the addition of antibiotics was required to eradicate remaining planktonic populations. In this study, a planktonic upregulated antigen was combined with the quadrivalent vaccine to remove the need for antibiotic therapy. Immunization with this pentavalent vaccine followed by intraperitoneal challenge of BALB/c mice with S. aureus resulted in 16.7% and 91.7% mortality in pentavalent vaccine and control groups, respectively (P < 0.001). Complete bacterial elimination was found in 66.7% of the pentavalent cohort, while only 8.3% of the control animals cleared the infection (P < 0.05). Further protective efficacy was observed in immunized rabbits following intramedullary challenge with S. aureus, where 62.5% of the pentavalent cohort completely cleared the infection, versus none of the control animals (P < 0.05). Passive immunization of BALB/c mice with serum IgG against the vaccine antigens prior to intraperitoneal challenge with S. aureus prevented mortality in 100% of mice and eliminated bacteria in 33.3% of the challenged mice. These results demonstrate that targeting both the planktonic and biofilm stages with the pentavalent vaccine or the IgG elicited by immunization can effectively protect against S. aureus infection.

Prosthetic joint infections are difficult to diagnose and treat due to biofilm formation by the causative pathogens. Pathogen identification relies on microbial culture that requires days to weeks, and in the case of chronic biofilm infections, lacks sensitivity. Diagnosis of infection is often delayed past the point of effective treatment such that only the removal of the implant is curative. Early diagnosis of an infection based on antibody detection might lead to less invasive, early interventions. Our study examined antibody-based assays against the Staphylococcus aureus biofilm-upregulated antigens SAOCOL0486 (a lipoprotein), glucosaminidase (a domain of SACOL1062), and SACOL0688 (the manganese transporter MntC) for detection of chronic S. aureus infection. We evaluated these antigens by enzyme-linked immunosorbent assay (ELISA) using sera from naive rabbits and rabbits with S. aureus-mediated osteomyelitis, and then we validated a proof of concept for the lateral flow assay (LFA). The SACOL0688 LFA demonstrated 100% specificity and 100% sensitivity. We demonstrated the clinical diagnostic utility of the SACOL0688 antigen using synovial fluid (SF) from humans with orthopedic implant infections. Elevated antibody levels to SACOL0688 in clinical SF specimens correlated with 91% sensitivity and 100% specificity for the diagnosis of S. aureus infection by ELISA. We found measuring antibodies levels to SACOL0688 in SF using ELISA or LFA provides a tool for the sensitive and specific diagnosis of S. aureus prosthetic joint infection. Development of the LFA diagnostic modality is a desirable, cost-effective option, potentially providing rapid readout in minutes for chronic biofilm infections.

Mycobacterium tuberculosis (MTB) causes both acute and chronic infections in humans characterized by tolerance to antibiotics and reactivation to cause secondary tuberculosis. These characteristics have led to renewed interested in the in vitro pellicle, or biofilm mode of growth, where bacteria grow to produce a thick aggregate at the air-liquid interface and exhibit increased phenotypic resistance to antibiotics. We infected guinea pigs with the virulent H37Rv strain of MTB for 60 days at which point we collected blood. To identify antigenic proteins, membrane protein extracts of MTB H37Ra pellicles and shaken cultures grown for 3, 5, or 7 weeks were probed with the infected animals’ sera after the proteins were separated by two-dimensional gel electrophoresis (2DGE). Antigenic proteins were then identified using MALDI-TOF/TOF mass spectrometry peptide mass fingerprinting. Antigenic pellicle proteins were compared across the three timepoints to identify those that were produced consistently during pellicle growth. They were also compared to those membrane proteins identified from harvested shaken cultures to determine pellicle-specific versus universally-expressed proteins. Using this technique we identified 44 distinct antigenic proteins, nine of which were pellicle-specific. The sequence of antigenic pellicle-specific proteins was checked for sequence conservation across 15 sequenced MTB clinical isolates, three other members of the MTB complex, as well as Mycobacterium avium and Mycobacterium smegmatis. The antigenic pellicle-specific protein Rv0097 was found to have very high sequence conservation within the MTB complex but not with related mycobacteria while FabG4 was highly conserved in all mycobacteria analyzed. These conserved pellicle-specific proteins represent targets for the development of future diagnostic tests and vaccines.

There is an urgent need to improve methods used to screen anti-tuberculosis drugs. An in vitro assay was developed to test drug treatment strategies that specifically target drug-tolerant Mycobacterium tuberculosis. The H37Rv strain of M. tuberculosis survived antimicrobial treatment as attached microbial communities when maintained in tissue culture media (RPMI-1640) with or without lysed human peripheral blood leukocytes. When cultured planktonically in the presence of Tween-80, bacilli failed to form microbial communities or reach logarithmic phase growth yet remained highly susceptible to antimicrobial drugs. In the absence of Tween, bacilli tolerated drug therapy by forming complex microbial communities attached to untreated well surfaces or to the extracellular matrix derived from lysed human leukocytes. Treatment of microbial communities with DNase I or Tween effectively dispersed bacilli and restored drug susceptibility. These data demonstrate that in vitro expression of drug tolerance by M. tuberculosis is linked to the establishment of attached microbial communities and that dispersion of bacilli targeting the extracellular matrix including DNA restores drug susceptibility. Modifications of this in vitro assay may prove beneficial in a high throughput platform to screen new anti-tuberculosis drugs especially those that target drug tolerant bacilli.

Objectives: Rhinosinusitis and polyposis are difficult to treat in patients with Samter's triad; they commonly recur despite sinus surgery, antibiotics, and/or nasal steroids. The present study assesses the efficacy of a multimodal regimen that includes topical corticosteroids and antibiotics delivered through a hydroxyethyl cellulose gel and by nebulization. Methods: Eleven patients with Samter's triad who had polyposis and rhinosinusitis that recurred despite endoscopic sinus surgery were treated with a 6-week course of multimodal topical therapy consisting of a hydroxyethyl cellulose gel that releases corticosteroids and antibiotics, topical nebulization of corticosteroids and antibiotics, saline solution rinses, and sinus debridement. Clinical outcomes were evaluated by Lund-Kennedy endoscopic and symptom scores. Histologic assessment was evaluated by hematoxylin and eosin staining before and after treatment. Results: Both Lund-Kennedy symptom and endoscopic scores showed a progressive and statistically significant decline throughout the course of treatment, reaching at 6 weeks 42% of the pretreatment values (p = 0.005) for the Lund-Kennedy symptom score and 34% (p = 0.002) for the endoscopic score, respectively; however, the significance of the improvement was lost with time. Conclusions: Topical gel therapy improves clinical symptoms, endoscopic findings, and sinus membrane histologic features in patients with refractory Samter's triad, but the improvement is transient, suggesting that a longer therapeutic period might be needed.

A new strategy employs amorphous melts of active pharmaceutical agents, which are formed with metal chlorides, including ZnCl2, through speciation ambiguity. The Lewis acidic, bioinorganic ionic liquid formulations improve shelf life and water solubility and eliminate issues related to crystal polymorphism. This broad-ranging approach produces thermally robust chlorometallate preparations for use as lotions, antibiofilm agents, and in situations without refrigeration.

Background: Chronic rhinosinusitis is a multifactorial disease resulting in impaired mucociliary clearance. Recent literature suggests that different bacterial species are associated with varied disease severity. We examined the immediate effect of microbial secreted factors on sinonasal ciliary function. Methods: Murine primary sinonasal cultures were established in an air-liquid interface (ALI). Bacterial supernatants were isolated from H. influenza, S. pneumoniae, S. aureus, and P. aeruginosa cultures, as well as co-cultures of H. influenza/S. pneumoniae and S. aureus/P. aeruginosa. Controlling for pH and osmolarity, supernatants were administered at 50% concentration to the apical surface of the ALI culture. Basal ciliary beat frequency (CBF) was recorded for 20 minutes, at 5-minute intervals. Control groups were treated with culture broth. At minimum, experiments were performed in triplicate. Stimulated CBF was recorded after mechanical stimulation via short bursts of pressurized air (55 mmHg). Results: All supernatants reduced basal CBF. S. pneumoniae and P. aeruginosa caused significant reduction in CBF at all time points, with the largest decrease of −46.3 ± 1.6% (p < 0.001) for S. pneumoniae and −27.1 ± 2.8% (p < 0.001) for P. aeruginosa. S. aureus caused the basal CBF to decline by −33.0 ± 2.8% (p < 0.001) at 5 minutes, which reversed by 20 minutes. Overall, H. influenza yielded the least change in CBF (−20.0 ± 2.8%, p < 0.002). Co-cultures (H. influenza/S. pneumoniae and S. aureus/P. aeruginosa) resulted in delayed CBF reduction compared with monocultures. P. aeruginosa also blunted stimulated CBF (p < 0.02). Conclusion: Results demonstrated acute decreases in murine sinonasal CBF after exposure to bacterial supernatants. Moreover, P. aeruginosa resulted in diminished ciliary stimulation capacity. © 2012 ARS-AAOA, LLC.

Silver carbenes may represent novel, broad-spectrum antimicrobial agents that have low toxicity while providing varying chemistry for targeted applications. Here, the bactericidal activity of four silver carbene complexes (SCCs) with different formulations, including nanoparticles (NPs) and micelles, was tested against a panel of clinical strains of bacteria and fungi that are the causative agents of many skin and soft tissue, respiratory, wound, blood, and nosocomial infections.MIC, MBC and multidose experiments were conducted against a broad range of bacteria and fungi. Time-release and cytotoxicity studies of the compounds were also carried out. Free SCCs and SCC NPs were tested against a panel of medically important pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Acinetobacter baumannii (MRAB), Pseudomonas aeruginosa, Burkholderia cepacia and Klebsiella pneumoniae.All four SCCs demonstrated strong efficacy in concentration ranges of 0.5-90 mg/L. Clinical bacterial isolates with high inherent resistance to purified compounds were more effectively treated either with an NP formulation of these compounds or by repeated dosing. Overall, the compounds were active against highly resistant bacterial strains, such as MRSA and MRAB, and were active against the biodefence pathogens Bacillus anthracis and Yersinia pestis. All of the medically important bacterial strains tested play a role in many different infectious diseases.The four SCCs described here, including their development as NP therapies, show great promise for treating a wide variety of bacterial and fungal pathogens that are not easily killed by routine antimicrobial agents.

Over the past 40 yr, epizootics of plague (Yersinia pestis) in northern Arizona have reduced populations of the Gunnison's prairie dog (Cynomys gunnisoni), with the exception of a large population found in the Aubrey Valley (AV). To examine potential mechanisms accounting for their survival, we collected prairie dog serum samples in 2005-2006 from AV and a neighboring population near Seligman (SE), Arizona. We quantified gene expression at 58 diverse immune proteins using a multiplexed enzyme-linked immunosorbent assay panel. We found a subset of proteins important in coagulation and inflammation (tissue factor [TF], calbindin [Cal], and thrombopoietin [TPO]) and T-cell responses (CD40L and CD40) that were present in AV at levels two to eight times greater than SE. These results suggest that AV and SE animals might differ in their ability to mount an immune response.

Staphylococcus aureus has reemerged as an important human pathogen in recent decades. Although many infections caused by this microbial species persist through a biofilm mode of growth, little is known about how the host's adaptive immune system responds to these biofilm infections. In this study, S. aureus cells adhered to pins in culture and were subsequently inserted into the tibiae of C57BL/6 mice, with an infecting dose of 2 × 10 5 CFU. This model was utilized to determine local cytokine levels, antibody (Ab) function, and T cell populations at multiple time points throughout infection. Like human hosts, S. aureus implant infection was chronic and remained localized in 100% of C57BL/6 mice at a consistent level of approximately 10 7 CFU/gram bone tissue after day 7. This infection persisted locally for >49 days and was recalcitrant to clearance by the host immune response and antimicrobial therapy. Local inflammatory cytokines of the Th1 (interleukin-2 [IL-2], IL-12 p70, tumor necrosis factor alpha [TNF-α], and IL-1β) and Th17 (IL-6 and IL-17) responses were upregulated throughout the infection, except IL-12 p70, which dwindled late in the infection. In addition, Th1 Ab subtypes against a biofilm antigen (SA0486) were upregulated early in the infection, while Th2 Abs and anti-inflammatory regulatory T cells (Tregs) were not upregulated until later. These results indicate that early Th1 and Th17 inflammatory responses and downregulated Th2 and Treg responses occur during the development of a chronic biofilm implant infection. This unrestrained inflammatory response may cause tissue damage, thereby enabling S. aureus to attach and thrive in a biofilm mode of growth.

Staphylococcus aureus infections, particularly those from methicillin-resistant strains (i.e., MRSA), are reaching epidemic proportions, with no effective vaccine available. The vast number and transient expression of virulence factors in the infectious course of this pathogen have made the discovery of protective antigens particularly difficult. In addition, the divergent planktonic and biofilm modes of growth with their accompanying proteomic changes also demonstrate significant hindrances to vaccine development. In this study, a multicomponent vaccine was evaluated for its ability to clear a staphylococcal biofilm infection. Antigens (glucosaminidase, an ABC transporter lipoprotein, a conserved hypothetical protein, and a conserved lipoprotein) were chosen since they were found in previous studies to have upregulated and sustained expression in a biofilm, both in vitro and in vivo . Antibodies against these antigens were first used in microscopy studies to localize their expression in in vitro biofilms. Each of the four antigens showed heterogeneous production in various locations within the complex biofilm community in the biofilm. Based upon these studies, the four antigens were delivered simultaneously as a quadrivalent vaccine in order to compensate for this varied production. In addition, antibiotic treatment was also administered to clear the remaining nonattached planktonic cells since the vaccine antigens may have been biofilm specific. The results demonstrated that when vaccination was coupled with vancomycin treatment in a biofilm model of chronic osteomyelitis in rabbits, clinical and radiographic signs of infection significantly reduced by 67 and 82%, respectively, compared to infected animals that were either treated with vancomycin or left untreated. In contrast, vaccination alone resulted in a modest, and nonsignificant, decrease in clinical (34% reduction) and radiographic signs (9% reduction) of infection, compared to nonvaccinated animal groups untreated or treated with vancomycin. Lastly, MRSA biofilm infections were significantly cleared in 87.5% of vaccinated and antibiotic-treated animals, while antibiotics or vaccine alone could not significantly clear infection compared to controls (55.6, 22.2, and 33.3% clearance rates, respectively). This approach to vaccine development may lead to the generation of vaccines against other pathogenic biofilm bacteria.

Chronic infection with the gram-negative organism Pseudomonas aeruginosa is a leading cause of morbidity and mortality in human patients, despite high doses of antibiotics used to treat the various diseases this organism causes. These infections are chronic because P. aeruginosa readily forms biofilms, which are inherently resistant to antibiotics as well as the host's immune system. Our laboratory has been investigating specific mutations in P. aeruginosa that regulate biofilm bacterial susceptibility to the host. To continue our investigation of the role of genetics in bacterial biofilm host resistance, we examined P. aeruginosa biofilms that lack the flgK gene. This mutant lacks flagella, which results in defects in early biofilm development (up to 36 h). For these experiments, the flgK -disrupted strain and the parental strain (PA14) were used in a modified version of the 96-well plate microtiter assay. Biofilms were challenged with freshly isolated human leukocytes for 4 to 6 h and viable bacteria enumerated by CFU. Subsequent to the challenge, both mononuclear cells (monocytes and lymphocytes) and neutrophils, along with tumor necrosis factor alpha (TNF-α), were required for optimal killing of the flgK biofilm bacteria. We identified a cytokine cross talk network between mononuclear cells and neutrophils that was essential to the production of lactoferrin and bacterial killing. Our data suggest that TNF-α is secreted from mononuclear cells, causing neutrophil activation, resulting in the secretion of bactericidal concentrations of lactoferrin. These results extend previous studies of the importance of lactoferrin in the innate immune defense against bacterial biofilms.

A series of N-heterocyclic carbene silver complexes have been synthesized and tested against the select group of bio-safety level 3 bacteria Burkholderia pseudomallei, Burkholderia mallei, Bacillus anthracis, methicillin-resistant Staphylococcus aureus and Yersinia pestis. Minimal inhibitory concentrations, minimal bactericidal and killing assays demonstrated the exceptional efficacy of the complexes against these potentially weaponizable pathogens.

Toxoplasma gondii often migrates to the central nervous system in immunocompromised patients, where it induces a severe inflammation referred to as Toxoplasma encephalitis. The mechanisms involved in control of parasite multiplication and prevention of Toxoplasma encephalitis remain unclear. The objective of the present study was to characterize the inflammatory response in the brains of mice during acute T. gondii infection, with emphasis on the expression of chemokine receptors. Susceptible C57BL/6 mice were orally infected with 10 cysts of the low-virulent ME49 strain of T. gondii. Levels of cytokines (TNF-alpha, IFN-gamma, IL-10, IL-6, and IL-12p70) and chemokines (CCL/2MCP-1) were measured in plasma at 5, 10, 15, 20, and 30 days after infection. In addition, the mRNA expression of chemokines (CCL5/RANTES, CCL2/MCP-1, CCL4/MIP-1beta) and chemokine receptors (CCR1, CCR2, CCR5, CCR7, CCR8, CXCR4, and CXR5) were measured in brain tissues at the same time points. Plasma levels of IFN-gamma and CCL2/MCP-1 were highly expressed at day 5, whereas TNF-alpha had a moderate increase at day 5, peaked at day 10, and returned to normal levels by day 30. Plasma levels of IL-10, IL-6, and IL-12p70 were not detected throughout the study. Analyses of mRNA expression of chemokines and chemokine receptors in the brain showed that CCL5/ RANTES, CCR7, CXCR4, and CXCR5 were upregulated, peaking after 10 days of T. gondii infection. IgM-specific antibody levels increased at day 5 and peaked at days 10 and 30, whereas IgG levels increased at day 10 and continued to increase thereafter, reaching maximum levels at day 30 postinfection (PI). Our results suggest that T. gondii infection is controlled at local and systemic levels, and that proinflammatory proteins and their receptors may be acting coordinately to induce stage conversion and prevent parasite multiplication and development of Toxoplasma encephalitis. The early production of IFN-gamma and the delayed expression of CXCR4 and CXCR5 indicate that T. gondii induces an early robust cellular immune response, followed by a strong and sustained antibody-mediated immunity.

Understanding the mechanisms implicated in the initial attachment, development, and maturation of a biofilm phenotype are of tremendous importance for their effect on the medical, industrial, and public health arenas. This review explores the current understanding of the nature of biofilms and the impact that molecular interactions between the bacteria themselves, as well as between bacteria and the host, may have on biofilm development and phenotype using the nonmotile Gram-positive coccus, Staphylococcus aureus, as an example.

Antigens from the methicillin-resistant Staphylococcus aureus (MRSA) cell wall have been shown to be immunogenic in vivo and upregulated during biofilm growth. In this study, we created purified, recombinant forms of selected antigens and biofilm-upregulated, cell wall-associated proteins. These proteins were shown to cause a robust polyclonal immunoglobulin G (IgG) response when used to immunize rabbits. Antibodies against these recombinant proteins bound to the native forms of each protein as harvested from in vitro grown biofilms of MRSA, as determined both via Western blot analysis and immunofluorescence confocal microscopy. These IgGs could be utilized as imaging tools that localize to areas of specific protein production within a biofilm. This work illustrates that immunogenic, cell wall-associated, biofilm-upregulated proteins are promising for in vitro visualization of biofilm growth, architecture, and space-function relationships.

Biofilms, communities of micro-organisms attached to a surface, are responsible for many chronic diseases and are often associated with environmental reservoirs or lifestyles. Bacillus anthracis is a Gram-positive, endospore-forming bacterium and is the aetiological agent of pulmonary, gastrointestinal and cutaneous anthrax. Anthrax infections are part of the natural lifecycle of many ruminants in North America, including cattle and bison, and B. anthracis is thought to be a central part of this ecosystem. However, in endemic areas in which humans and livestock interact, chronic cases of cutaneous anthrax are commonly reported. This suggests that biofilms of B. anthracis exist in the environment and are part of the ecology associated with its lifecycle. Currently, there are few data that account for the importance of the biofilm mode of life in B. anthracis, yet biofilms have been characterized in other pathogenic and non-pathogenic Bacillus species, including Bacillus cereus and Bacillus subtilis, respectively. This study investigated the phenotypic and functional role of biofilms in B. anthracis. The results demonstrate that B. anthracis readily forms biofilms which are inherently resistant to commonly prescribed antibiotics, and that antibiotic resistance is not solely the function of sporulation.

Osteomyelitis is a progressive infection of bone, that results in inflammatory destruction of the bone, bone necrosis, and new bone formation and may progress to a chronic and persistent state. The major categories of osteomyelitis are based on the source of infection (hematogenous or secondary to a contiguous focus of infection) and whether vascular insufficiency (either local or systemic) exists. While large-organism inoculation and/or host compromise can predispose patients to the development of osteomyelitis, the virulence of the infecting pathogen also has a significant role. One species in particular, Staphylococcus aureus , is able to cause an acute bone infection even with a low inoculum in a healthy host. In addition, through the timed expression of its arsenal of virulence factors and aided by its ability to develop antibiotic resistance rapidly, S. aureus progresses to a chronic, biofilm-mediated infection. Once a chronic infection develops, bacterial clearance cannot be attained by the host immune system or antimicrobial therapy. At this point, surgical removal of the nidus of infection is usually necessary for complete infection resolution.

Biofilms have been found on the inner surface of silicone tubing inside dialysis machines. Endotoxin releasing from those biofilms increases the bioincompatibility of dialysis liquids and leads to long-term inflammatory complications among dialysis patients. Endotoxin measurement is recommended for the control of dialysis liquids. This article describes the use of a new method, the Whole Blood Assay (WBA), for endotoxin quantification in dialysis biofilms. Biofilms were suspended in sterile water by scraping the tubing samples. Diluted blood samples from healthy donors were stimulated overnight with the contaminated suspension. Stimulated mononuclear cells released IL-1beta in response to endotoxins. IL-1beta level was then measured using an ultrasensitive ELISA method. We demonstrated a semilogarithmic model in which the optical densities measured after the ELISA assay increases linearly with the levels of endotoxin. This model allowed the determination of the amount of endotoxins in biofilm samples with a detection limit of 0.032 EU/mL. Most of the time, the amounts of endotoxin measured by the WBA were higher than those measured by the Limulus Amoebocyte Lysate (LAL) assay. This study suggested the presence of "endotoxin-like" compounds different from the lipopolysaccharides that are not detected by the LAL assay. We concluded that the LAL is necessary but insufficient to have a representative quantification of endotoxins that could be hazardous to patient health.

Objective: To evaluate the clinical effectiveness of rhinotopic therapy, and its impact on the mucosal membrane and mucosal biofilms of patients with refractory chronic rhinosinusitis (CRS). Study design: Prospective pilot clinical study. Setting: Tertiary rhinology referral center. Subjects and methods: Twenty-three patients with refractory CRS were treated with the rhinotopic protocol, a multimodality therapy consisting of topical sinus application of antibiotics and corticosteroids through gel and nebulization, endoscopic sinus debridement and saline irrigation. Clinical outcome was assessed using Lund-Kennedy (LK) symptom and endoscopic appearance scores. Mucosal epithelial barrier changes were evaluated through H & E stain. The impact on biofilms was assessed through colony forming units (CFUs) counts. Results: Following treatment, there was a significant improvement in the patients’ LK symptom and endoscopic appearance scores, in the mucosal epithelial barrier and in the biofilms CFUs count. The clinical improvement was sustained at least 6 months following completion of therapy. Conclusion: Refractory CRS is a difficult to treat entity that does not respond to traditional medical therapy, including repeated oral antibiotics, commercial nasal steroids, nasal saline rinses and sinus debridement. This work shows that rhinotopic therapy is effective in improving clinical symptoms, healing mucosal damage and disrupting biofilm load in refractory CRS patients. Further work is needed to investigate if single aspects or a collective implementation of the different components of this therapy are responsible for improvement.

Staphylococcus aureus is a common pathogen responsible for nosocomial and community infections. It readily colonizes indwelling catheters, forming microbiotic communities termed biofilms. S. aureus bacteria in biofilms are protected from killing by antibiotics and the body's immune system. For years, one mechanism behind biofilm resistance to attack from the immune system's sentinel leukocytes has been conceptualized as a deficiency in the ability of the leukocytes to penetrate the biofilm. We demonstrate here that under conditions mimicking physiological shear, leukocytes attach, penetrate, and produce cytokines in response to maturing and fully matured S. aureus biofilm.

Several monoclonal antibodies derived from hybridomas from mice that had been immunized with recombinant Mycobacterium bovis antigen 85 (Ag85) were tested for reactivity against antigen 85 (Ag85) from M. bovis and against sera from 100 bison inoculated with M. paratuberculosis and from 100 control bison from a disease-free herd. Monoclonal antibodies mAb85.1, mAb85.44.1, mAb85.44.9, and mAb85.96 reacted against three or four 30-33-kDa bands of the Ag85 complex of M. bovis. Importantly, these mAbs also reacted with bands of similar molecular weight in the sera of bison inoculated with M. paratuberculosis. Additionally, when sera from 198 bison in four herds were reacted against mAb85.1 and mAb85.96, 26 bison reacted positively for the presence of Ag85 by either mAb or by both. These preliminary results indicate that monoclonal antibodies may eventually lead to a reliable diagnostic test for the early detection of M. paratuberculosis infections in ruminants as well as to a means for identifying contaminated dairy products.

Endophthalmitis is an important disease of the eye that is most frequently caused by postoperative and post-traumatic introduction of bacteria into the posterior segment of the eye. In the case of severe infections, visual acuity is greatly damaged or completely lost. Much work has focused on the ability of planktonic bacteria to cause infection and ocular damage while little work has focused on chronic infections in endophthalmitis mediated by the formation of bacterial biofilms on the surface of the lens. This review focuses on the interaction of Staphylococcus aureus and Staphylococcus epidermidis lens-associated biofilms in endophthalmitis. Additionally, this review highlights some relevant biofilm-immune system interactions and outlines a new in vivo mouse model to explore biofilm-related infections in endophthalmitis.

L-selectin is constitutively expressed on most leukocytes and is responsible for the initial events in cell trafficking termed tethering and rolling. Recently, L-selectin has been shown to associate with the actin-based cytoskeleton under a variety of conditions. In an effort to better understand L-selectin cytoskeletal association and the ultrastructural nature of the cytoskeleton itself, we provide a comparison of the cytoskeletal association of various human and bovine surface proteins in relation to L-selectin. Electron microscopic examination of the cytoskeleton provided further data on the ultrastructure of freshly isolated peripheral lymphocytes as well as demonstrated L-selectin localization to the periphery of the cytoskeleton following low dose detergent treatment of the cell. Clusters of colloidal-gold-stained L-selectin were found on the surface of the detergent-treated lymphocytes, even though these particles completely lacked microvilli. By flow cytometry, we have defined three distinct patterns of cytoskeletal association; constitutive, inductive, and mAb crosslink-induced, and assigned human and bovine CD2, CD3, CD4, CD5, CD8, CD18, CD19, CD44, CD45RA, CD45RO, alphabeta TCR, gammadelta TCR, E-selectin ligands, and L-selectin surface antigens to one of these respective patterns. SDS-PAGE analyses confirmed most of the flow cytometry results. Depending upon its conformation, L-selectin fell into the inductive or mAb crosslink-induced pattern of association, similar to E-selectin ligand(s). Our data provide additional insight into the functional role of L-selectin and the cytoskeleton in immunological events.

L-Selectin mediates leukocyte rolling on endothelium and immobilized leukocytes. Its regulation has been the subject of much study, and the conformation of the molecule may play an important role in its function. Here we report that a conformational change in L-selectin, induced by an anti-lectin domain mAb (LAM1-116) and recognized by another mAb directed to a conserved epitope on L-selectin (EL-246), predisposed L-selectin to cytoskeletal association. This effect was due to direct binding of the mAb, not to overt signaling events, and was specific to LAM1-116. Nineteen other anti-L-selectin mAbs directed against the lectin, epidermal growth factor, or short consensus repeat domains lacked this activity. The induced conformational change occurred at 37°C, at 4°C, in the presence of sodium azide and tyrosine kinase inhibitors herbimycin A and genistein, and with soluble detergent-extracted L-selectin. In the presence of LAM1-116, EL-246 induced cytoskeletal association of L-selectin in the absence of Ab cross-linking as visualized by L-selectin staining after low dose detergent treatment of the cells. We propose that the conformational change described herein regulates L-selectin-mediated events by exposing a high avidity binding site that, when engaged, triggers association of L-selectin with the cytoskeleton, which may lead to stronger tethers with physiological ligands.

Compared to most alphabeta T cells, bovine gammadelta T cells express two to five times the level of L-selectin and have a much higher capacity to roll on monolayers of endothelial cells, platelets, and leukocytes in assays done under physiological flow. To gain additional insight into the basis for these differences, scanning (SEM) and transmission (TEM) electron microscopy were used to compare the cellular ultrastructure of bovine gammadelta and alphabeta T cells and to study the expression of L-selectin on these cells. It is interesting that gammadelta T cells had more than twice as many microvilli and other surface projections on a per cell basis as alphabeta T cells. This was not due to the gammadelta T cell being larger; after adhesion and fixation procedures used for the EM studies, gammadelta T cells averaged 3.9 +/- 0.01 microm in diameter, whereas alphabeta T cells were slightly larger (5.7 +/- 0.01 microm in diameter). As previously shown for human neutrophils and lymphocytes, L-selectin was preferentially localized to the tips of the microvilli. In contrast, WC1, a lineage-specific antigen on gammadelta T cells, was localized on the plasmalemma between the microvilli. Our findings suggest that more effective rolling of gammadelta T cells in various in vitro flow assays may be due to the greater number of microvilli on gammadelta T cells leading to a higher number of contact sites during adhesion events. In addition, this physical parameter may explain the increased level of L-selectin expression on gammadelta versus alphabeta T cells because L-selectin is clustered at the tips of microvilli.

Fosmidomycin is a time-dependent nanomolar inhibitor of methylerythritol phosphate (MEP) synthase, which is the enzyme that catalyzes the first committed step in the MEP pathway to isoprenoids. Importantly, fosmidomycin is one of only a few MEP pathway-specific inhibitors that exhibits antimicrobial activity. Most inhibitors identified to date only exhibit activity against isolated pathway enzymes. The MEP pathway is the sole route to isoprenoids in many bacteria, yet has no human homologs. The development of inhibitors of this pathway holds promise as novel antimicrobial agents. Similarly, analyses of the bacterial response toward MEP pathway inhibitors provides valuable information toward the understanding of how emergent resistance may ultimately develop to this class of antibiotics. We have examined the transcriptional response of Salmonella enterica serovar typhimurium LT2 to sub-inhibitory concentrations of fosmidomycin via cDNA microarray and RT-PCR. Within the regulated genes identified by microarray were a number of genes encoding enzymes associated with the mediation of reactive oxygen species (ROS). Regulation of a panel of genes implicated in the response of cells to oxidative stress (including genes for catalases, superoxide dismutases, and alkylhydrogen peroxide reductases) was investigated and mild upregulation in some members was observed as a function of fosmidomycin exposure over time. The extent of regulation of these genes was similar to that observed for comparable exposures to kanamycin, but differed significantly from tetracycline. Furthermore, S. typhimurium exposed to sub-inhibitory concentrations of fosmidomycin displayed an increased sensitivity to exogenous H2O2 relative to either untreated controls or kanamycin-treated cells. Our results suggest that endogenous oxidative stress is one consequence of exposures to fosmidomycin, likely through the temporal depletion of intracellular isoprenoids themselves, rather than other mechanisms that have been proposed to facilitate ROS accumulation in bacteria (e.g. cell death processes or the ability of the antibiotic to redox cycle).

Objectives The purpose of this study is to characterize the association between altered epithelial barrier function, represented by changes in histology and differential expression of the mucosal water membrane permeability protein aquaporin 5 (AQP5), and the pathophysiology of chronic refractory sinusitis (CRS) in patients with and without nasal polyposis. Study design Prospective clinical study. Setting Tertiary rhinology referral center. Participants Sinonasal samples were obtained from seven CRS subjects with nasal polyps (CRSwNP), seven CRS without nasal polyposis (CRSsNP), and five control healthy patients. Methods Mucosal membrane changes were evaluated through hematoxylin and eosin staining of the membrane barrier and immunohistochemical staining of AQP5 expression, a membrane channel protein that affects trans-epithelial water permeability and tissue edema. AQP5 expression was confirmed by real-time PCR (rt-PCR) and western blot. Levels of other membrane proteins, including E-cadherin and Septin-2, were also assessed. Results CRSwNP patients showed substantial histologic evidence of membrane remodeling with increased edema and glandular hyperplasia. The epithelial expression of AQP5 was significantly lower in CRSwNP as compared to CRSsNP or control. There was no significant difference in the expression of E-cadherin and Septin-2. Conclusions Collectively, these data suggest that the mucosal epithelial barrier is compromised in the context of CRS (predominantly in CRSwNP) when compared to control and that AQP5 acts as a key tight junction protein in the maintenance of mucosal water homeostasis. We hypothesize that AQP5 plays a possible role in the pathophysiology of mucosal edema and polyp formation.

Chronic rhinosinusitis (CRS) is a common inflammatory disease of the sinonasal cavity mediated, in part, by polymicrobial communities of bacteria. Recent molecular studies have confirmed the importance of Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi) in CRS. Here, we hypothesize that interaction between S. pneumoniae and NTHi mixed-species communities cause a change in bacterial virulence gene expression. We examined CRS as a model human disease to validate these polymicrobial interactions. Clinical strains of S. pneumoniae and NTHi were grown in mono- and co-culture in a standard biofilm assay. Reverse transcriptase real-time PCR (RTqPCR) was used to measure gene expression of key virulence factors. To validate these results, we investigated the presence of the bacterial RNA transcripts in excised human tissue from patients with CRS. Consequences of physical or chemical interactions between microbes were also investigated. Transcription of NTHi type IV pili was only expressed in co-culture in vitro, and expression could be detected ex vivo in diseased tissue. S. pneumoniae pyruvate oxidase was up-regulated in co-culture, while pneumolysin and pneumococcal adherence factor A were down-regulated. These results were confirmed in excised human CRS tissue. Gene expression was differentially regulated by physical contact and secreted factors. Overall, these data suggest that interactions between H. influenzae and S. pneumoniae involve physical and chemical mechanisms that influence virulence gene expression of mixed-species biofilm communities present in chronically diseased human tissue. These results extend previous studies of population-level virulence and provide novel insight into the importance of S. pneumoniae and NTHi in CRS.

Staphylococcus aureus is a common cause of prosthetic implant infections, which can become chronic due to the ability of S. aureus to grow as a biofilm. Little is known about adaptive immune responses to these infections in vivo . We hypothesized that S. aureus elicits inflammatory Th1/Th17 responses, associated with biofilm formation, instead of protective Th2/Treg responses. We used an adapted mouse model of biofilm-mediated prosthetic implant infection to determine chronic infection rates, Treg cell frequencies, and local cytokine levels in Th1-biased C57BL/6 and Th2-biased BALB/c mice. All C57BL/6 mice developed chronic S. aureus implant infection at all time points tested. However, over 75% of BALB/c mice spontaneously cleared the infection without adjunctive therapy and demonstrated higher levels of Th2 cytokines and anti-inflammatory Treg cells. When chronic infection rates in mice deficient in the Th2 cytokine interleukin-4 (IL-4) via STAT6 mutation in a BALB/c background were assessed, the mice were unable to clear the S. aureus implant infection. Additionally, BALB/c mice depleted of Treg cells via an anti-CD25 monoclonal antibody (MAb) were also unable to clear the infection. In contrast, the C57BL/6 mice that were susceptible to infection were able to eliminate S. aureus biofilm populations on infected intramedullary pins once the Th1 and Th17 responses were diminished by MAb treatment with anti-IL-12 p40. Together, these results indicate that Th2/Treg responses are mechanisms of protection against chronic S. aureus implant infection, as opposed to Th1/Th17 responses, which may play a role in the development of chronic infection.

Increasing attention has been focused on understanding bacterial biofilms and this growth modality's relation to human disease. In this review we explore the genetic regulation and molecular components involved in biofilm formation and maturation in the context of the Gram-positive cocci, Staphylococcus aureus. In addition, we discuss diseases and host immune responses, along with current therapies associated with S. aureus biofilm infections and prevention strategies.

There is mounting evidence that bacterial and possibly fungal biofilms play an important role in the etiology and persistence of Chronic Rhinosinusitis (CRS). CRS affects nearly 16–25% of the US population each year, with billions of dollars of annual healthcare expenditures dedicated to its treatment (Gliklich and Metson 1995). Unfortunately, the recalcitrant nature of the disease, which often exhibits a chronic relapsing course, significantly contributes to these healthcare costs. The reasons for the persistent nature of the disease are likely secondary to a number of underlying pathophysiologic mechanisms. Asthma, allergic rhinitis, Gram-positive and Gram-negative infections, aspirin-sensitive asthma, fungus, osteitis, nasal polyposis, superantigens, and other factors have been implicated as etiologies contributing to the development of CRS.

The fungal species Candida albicans and the bacterial species Staphylococcus aureus are responsible for a majority of hospital-acquired infections and often coinfect critically ill patients as complicating polymicrobial biofilms. To investigate biofilm structure during polymicrobial growth, dual-species biofilms were imaged with confocal scanning laser microscopy. Analyses revealed a unique biofilm architecture where S. aureus commonly associated with the hyphal elements of C. albicans. This physical interaction may provide staphylococci with an invasion strategy because candidal hyphae can penetrate through epithelial layers. To further understand the molecular mechanisms possibly responsible for previously demonstrated amplified virulence during coinfection, protein expression studies were undertaken. Differential in-gel electrophoresis identified a total of 27 proteins to be significantly differentially produced by these organisms during coculture biofilm growth. Among the upregulated staphylococcal proteins was l-lactate dehydrogenase 1, which confers resistance to host-derived oxidative stressors. Among the downregulated proteins was the global transcriptional repressor of virulence factors, CodY. These findings demonstrate that the hyphae-mediated enhanced pathogenesis of S. aureus may not only be due to physical interactions but can also be attributed to the differential regulation of specific virulence factors induced during polymicrobial growth. Further characterization of the intricate interaction between these pathogens at the molecular level is warranted, as it may aid in the design of novel therapeutic strategies aimed at combating fungal–bacterial polymicrobial infection.

Background Bacterial biofilms consist of a complex, organized community of bacteria that anchor to both biotic and abiotic surfaces. They are composed of layers of embedded, live bacteria within extruded exopolymeric matrix. This configuration allows for evasion of host defenses and decreased susceptibility to antibiotic therapy while maintaining the ability to deliberately release planktonic bacteria, resulting in recurrent acute infections. Thus, bacterial biofilms were hypothesized to contribute to the progression and persistence of chronic rhinosinusitis. Methods This review summarizes several of the seminal papers supporting this hypothesis. Results Multiple reports using various imaging modalities have demonstrated the presence of biofilms in sinonasal mucosa of patients with chronic rhinosinusitis. More recently, several studies have correlated the presence of biofilms with poor clinical outcomes in the disease process. Early therapeutic interventions have generated mixed results. Conclusions Bacterial biofilms appear to contribute to the progression of chronic rhinosinusitis in a subset of patients, although substantial effort toward therapeutic intervention is still necessary.

The cover picture is a composite design depicting the variety in NHC complex chemistry. Like pieces in a puzzle, all details contribute to our growing knowledge of these compounds. The first puzzle piece in the bottom left corner represents the carbene precursors, essential in all synthesis design (C. W. Bielawski et al.; p. 1729 ff). The background reflects the importance of these complexes, here in the homogeneous catalytic chemistry of gaseous molecules (C. M. Crudden et al.; p. 1746 ff), while the diverse structural types are highlighted by the zwitterionic “crabenes” mid right (L. Delaude; p. 1681 ff). Related aspects such as the physical chemistry or biological relevance are not forgotten: the sketch of the buried volume of an NHC ligand symbolizes the former (L. Cavallo et al.; p. 1759 ff), and the antimicrobial activity of silver–NHC complexes against biosafety level 3 bacteria typifies the latter (W. J. Youngs et al.; p. 1739 ff). Mr. Francois Nicks is acknowledged for drawing the “crabene”. The editorial office thanks all authors for their excellent contributions to this cover picture puzzle. Finally, there are many gaps. EurJIC hopes that this issue will stimulate you to discover another fragment that will complete the picture.

Biofilm infections are a therapeutic and diagnostic challenge due to their location and persistence. Specimen culturing is the golden standard for diagnosing bacterial infections, but recent investigation of the ability of this technique to grow bacteria from a biofilm has indicated that it is not reliable. Other testing modalities, such as PCR and serology assays, are either nonspecific for biofilm infections or they include the risk of contamination during sampling. Therefore, accurate diagnosis of an infection usually takes days and requires extensive test procedures, leading to increased healthcare costs and discomfort to the patient. In recent years, many attempts have been performed to design and set up new serology diagnostic assays to obtain early, noninvasive diagnosis of infections sustained by biofilms colonizing native tissues and medical implants. This chapter describes such attempts, including some examples of tests that could eliminate many of the obstacles related to early diagnosis in biofilm infection. The new tools could also allow new medical and surgical approaches to monitor and treat biofilm infections associated with many medical implants.

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

Prosthetic implantation is rising in popularity in the United States, and use of prostheses will only continue to increase with the aging of the Baby Boomer generation. A concurrent rise in the number of infections of these implants is also being seen. Prosthetic implant infection (PII) can be caused by direct inoculation of bacteria to the implant or by seeding from the blood (hematogenous). Because the implant is quickly coated by host connective tissue upon implantation, bacteria such as the staphylococcal species S. aureus and S. epidermidis are able to readily gain a foothold in the host. S. aureus is able to quickly develop antibiotic resistance and methicillin-resistant strains (MRSA) are considered endemic in hospitals. This bacterium features a myriad of virulence factors that allow it to colonize and damage the host, as well as avoid the host immune response. These virulence factors are largely regulated through population-based quorum sensing via the agr system. S. aureus is also able to form biofilms, microbial communities encased in a polysaccharide matrix, which allows the bacteria within to persist in the face of antimicrobial therapies and the host response. Diagnosis of PII is difficult with many false results and confusion with aseptic loosening of the implant. Because of ineffective means of diagnosis, combined with S. aureus’ high incidence of antibiotic resistance and its ability to evade both antibiotics and the host response through biofilm formation, treatment for PII is often inadequate and infection can become chronic. Therefore, PII has a high rate of morbidity and mortality for patients, as well as an extreme economic burden on the US healthcare system.

Staphylococcus aureus causes persistent, recurrent infections (e.g., osteomyelitis) by forming biofilms. To survey the antibody-mediated immune response and identify those proteins that are immunogenic in an S. aureus biofilm infection, the tibias of rabbits were infected with methicillin-resistant S. aureus to produce chronic osteomyelitis. Sera were collected prior to infection and at 14, 28, and 42 days postinfection. The sera were used to perform Western blot assays on total protein from biofilm grown in vitro and separated by two-dimensional gel electrophoresis. Those proteins recognized by host antibodies in the harvested sera were identified via matrix-assisted laser desorption ionization-time of flight analysis. Using protein from mechanically disrupted total and fractionated biofilm protein samples, we identified 26 and 22 immunogens, respectively. These included a cell surface-associated β-lactamase, lipoprotein, lipase, autolysin, and an ABC transporter lipoprotein. Studies were also performed using microarray analyses and confirmed the biofilm-specific up-regulation of most of these genes. Therefore, although the biofilm antigens are recognized by the immune system, the biofilm infection can persist. However, these proteins, when delivered as vaccines, may be important in directing the immune system toward an early and effective antibody-mediated response to prevent chronic S. aureus infections. Previous works have identified S. aureus proteins that are immunogenic during acute infections, such as sepsis. However, this is the first work to identify these immunogens during chronic S. aureus biofilm infections and to simultaneously show the global relationship between the antigens expressed during an in vivo infection and the corresponding in vitro transcriptomic and proteomic gene expression levels.

The research results presented here are part of a more extensive effort regarding sustained bioconvection in porous media. Bioconvection is the phenomenon of gravity driven fluid motion due to buoyancy forces resulting from density differences between the fluid and motile micro-organisms suspended in the fluid. While the field of bio-convection in pure fluids emerged substantially over the past decade the corresponding effects of bio-convection in porous media received much less attention, despite the fact that micro-organisms grow naturally in porous environments; soil, food and human tissues serve as basic examples. The research focuses in two major new directions. The first deals with the theoretical and experimental investigation of bio-convection in porous media. The second major new direction is linked to the sustainability of the bio-convection motion. The existing work on bio-convection in both pure fluids and porous media exclude micro-organism growth during the bio-convection because the time scales concerned were very short. However, when the question of the sustainability of this convection over long times arises, microorganism growth has to be accounted for. If sustained bio-convection in porous media is possible it opens the avenue to investigate its impact on microbial proliferation in soil, food and human tissue, an important avenue for application of the theoretical results. Then, if bio-convection enhances microbial proliferation it may be undesirable in some cases, e.g. in food, or it might be desirable if specific micro-organisms that can be used for contaminated soil remediation will be "helped" by the bio-convection process to access contaminated regions in the soil. The theoretical and experimental results presented in this paper reflect the process of monotonic growth of motile microorganisms (e.g. the PAOI strain of Pseudomonas Aeruginosa) to be included in the bioconvection process. A new proposed model is shown to be the appropriate one to better reflect both conceptually as well as practically the microbial growth process.