Auto Poisoning of the Respiratory Chain by a Quorum-Sensing-Regulated Molecule Favors Biofilm Formation and Antibiotic Tolerance.

Bacterial programmed cell death and quorum sensing are direct examples of prokaryote group behaviors, wherein cells coordinate their actions to function cooperatively like one organism for the benefit of the whole culture. We demonstrate here that 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO), a Pseudomonas aeruginosa quorum-sensing-regulated low-molecular-weight excreted molecule, triggers autolysis by self-perturbing the electron transfer reactions of the cytochrome bc1 complex. HQNO induces specific self-poisoning by disrupting the flow of electrons through the respiratory chain at the cytochrome bc1 complex, causing a leak of reducing equivalents to O2 whereby electrons that would normally be passed to cytochrome c are donated directly to O2. The subsequent mass production of reactive oxygen species (ROS) reduces membrane potential and disrupts membrane integrity, causing bacterial cell autolysis and DNA release. DNA subsequently promotes biofilm formation and increases antibiotic tolerance to beta-lactams, suggesting that HQNO-dependent cell autolysis is advantageous to the bacterial populations. These data identify both a new programmed cell death system and a novel role for HQNO as a critical inducer of biofilm formation and antibiotic tolerance. This newly identified pathway suggests intriguing mechanistic similarities with the initial mitochondrial-mediated steps of eukaryotic apoptosis.

Antifungal activity against planktonic and biofilm Candida albicans in an experimental model of foreign-body infection.

OBJECTIVES: The treatment of Candida implant-associated infections remains challenging. We investigated the antifungal activity against planktonic and biofilm Candida albicans in a foreign-body infection model. METHODS: Teflon cages were subcutaneously implanted in guinea pigs, infected with C. albicans (ATCC 90028). Animals were treated intraperitoneally 12 h after infection for 4 days once daily with saline, fluconazole (16 mg/kg), amphotericin B (2.5 mg/kg), caspofungin (2.5 mg/kg) or anidulafungin (20 mg/kg). Planktonic Candida was quantified, the clearance rate and cure rate determined. RESULTS: In untreated animals, planktonic Candida was cleared from cage fluid in 25% (infected with 4.5 × 10(3) CFU/cage), 8% (infected with 4.8 × 10(4) CFU/cage) and 0% (infected with 6.2 × 10(5) CFU/cage). Candida biofilm persisted on all explanted cages. Compared to untreated controls, caspofungin reduced the number of planktonic C. albicans to 0.22 and 0.0 CFU/ml, respectively, and anidulafungin to 0.11 and 0.13 CFU/ml, respectively. Fluconazole cured 2/12 cages (17%), amphotericin B and anidulafungin 1/12 cages (8%) and caspofungin 3/12 cages (25%). CONCLUSION: Echinocandins showed superior activity against planktonic C. albicans. Caspofungin showed the highest cure rate of C. albicans biofilm. However, no antifungal exceeded 25% cure rate, demonstrating the difficulty of eradicating Candida biofilms from implants.

Apical access and closure devices for transapical transcatheter heart valve procedures.

The majority of transcatheter aortic valve implantations, structural heart procedures and the newly developed transcatheter mitral valve repair and replacement are traditionally performed either through a transfemoral or a transapical access site, depending on the presence of severe peripheral vascular disease or anatomic limitations. The transapical approach, which carries specific advantages related to its antegrade nature and the short distance between the introduction site and the cardiac target, is traditionally performed through a left anterolateral mini-thoracotomy and requires rib retractors, soft tissue retractors and reinforced apical sutures to secure, at first, the left ventricular apex for the introduction of the stent-valve delivery systems and then to seal the access site at the end of the procedure. However, despite the advent of low-profile apical sheaths and newly designed delivery systems, the apical approach represents a challenge for the surgeon, as it has the risk of apical tear, life-threatening apical bleeding, myocardial damage, coronary damage and infections. Last but not least, the use of large-calibre stent-valve delivery systems and devices through standard mini-thoracotomies compromises any attempt to perform transapical transcatheter structural heart procedures entirely percutaneously, as happens with the transfemoral access site, or via a thoracoscopic or a miniaturised video-assisted percutaneous technique. During the past few years, prototypes of apical access and closure devices for transapical heart valve procedures have been developed and tested to make this standardised successful procedure easier. Some of them represent an important step towards the development of truly percutaneous transcatheter transapical heart valve procedures in the clinical setting.

Role of Aeromonas hydrophila flagella glycosylation in adhesion to Hep-2 cells, biofilm formation and immune stimulation.

Polar flagellin proteins from Aeromonas hydrophila strain AH-3 (serotype O34) were found to be O-glycosylated with a heterogeneous heptasaccharide glycan. Two mutants with altered (light and strong) polar flagella glycosylation still able to produce flagella were previously obtained, as well as mutants lacking the O34-antigen lipopolysaccharide (LPS) but with unaltered polar flagella glycosylation. We compared these mutants, altogether with the wild type strain, in different studies to conclude that polar flagella glycosylation is extremely important for A. hydrophila adhesion to Hep-2 cells and biofilm formation. Furthermore, the polar flagella glycosylation is an important factor for the immune stimulation of IL-8 production via toll receptor 5 (TLR5).

The influence of substratum type and nutrient supply on biofilm organic matter utilization in streams

We investigated the effect of benthic substratum type (sand and rocks) and nutrient supply (N and P) on biofilm structure and heterotrophic metabolism in a field experiment in a forested Mediterranean stream (Fuirosos). Rock and sand colonization and biofilm formation was intensively studied for 44 d at two stream reaches: control and experimental (continuous addition of phosphate, ammonia, and nitrate). Structural (C, N, and polysaccharide content and bacterial and chlorophyll density) and metabolic biofilm parameters (b-glucosidase, peptidase, and phosphatase enzyme activities) were analyzed throughout the colonization process. The epilithic biofilm (grown on rocks) had a higher peptidase activity at the impacted reach, together with a higher algal and bacterial biomass. The positive relationship between the peptidase activity per cell and the N content of the epilithic biofilm suggested that heterotrophic utilization of proteinaceous compounds from within the biofilm was occurring. In contrast, nutrient addition caused the epipsammic biofilm (grown on sand) to exhibit lower b-glucosidase and phosphatase activities, without a significant increase in bacterial and algal biomass. The differential response to nutrient addition was related to different structural characteristics within each biofilm. The epipsammic biofilm had a constant and high C:N ratio (22.7) throughout the colonization. The epilithic biofilm had a higher C:N ratio at the beginning of the colonization (43.2) and evolved toward a more complex structure (high polysaccharide content and low C:N ratio) during later stages. The epipsammic biofilm was a site for the accumulation and degradation of organic matter: polysaccharides and organic phosphorus compounds had higher degradation activities

The dynamics of biofilm bacterial communities is driven by flow wax and wane in a temporary stream

Biofilm communities are exposed to long periods of desiccation in temporary streams. We investigated how water flow intermittency affected the bacterial community structure colonizing three different streambed compartments in a Mediterranean stream. Massive parallel sequencing revealed different bacterial communities in biofilms from sand sediments and cobbles. Bacterial communities were similar (62% of shared operational taxonomic units) in the epipsammic and hyporheic biofilms, and more diverse than those in the epilithic biofilms. The non-flow phase caused a decrease of bacterial diversity in the biofilms, when communities included only bacterial taxa assumed to be adapted to water stress. The most sensitive bacterial communities to flow intermittency were in the epilithic, where the exposure to physical stress was the highest. In sand sediments a wide group of bacterial taxa was tolerant to desiccation. During non-flow the proliferation of opportunistic taxa in the superficial compartments evidenced the biological link with the terrestrial environment. Bacterial communities better tolerate rewetting than desiccation, since a major number of taxa tolerant to rewetting occurred in all biofilms. Overall, bacterial communities in sandy compartments showed higher resistance to flow intermittency than those in epilithic biofilms

Emerging, non-PCV13 serotypes 11A and 35B of Streptococcus pneumoniae show high potential for biofilm formation in vitro

Background: Since the use of pneumococcal conjugate vaccines PCV7 and PCV13 in children became widespread, invasive pneumococcal disease (IPD) has dramatically decreased. Nevertheless, there has been a rise in incidence of Streptococcus pneumoniae non-vaccine serotypes (NVT) colonising the human nasopharynx. Nasopharyngeal colonisation, an essential step in the development of S. pneumoniae-induced IPD, is associated with biofilm formation. Although the capsule is the main pneumococcal virulence factor, the formation of pneumococcal biofilms might, in fact, be limited by the presence of capsular polysaccharide (CPS). Methodology/Principal Findings: We used clinical isolates of 16 emerging, non-PCV13 serotypes as well as isogenic transformants of the same serotypes. The biofilm formation capacity of isogenic transformants expressing CPSs from NVT was evaluated in vitro to ascertain whether this trait can be used to predict the emergence of NVT. Fourteen out of 16 NVT analysed were not good biofilm formers, presumably because of the presence of CPS. In contrast, serotypes 11A and 35B formed >45% of the biofilm produced by the non-encapsulated M11 strain. Conclusions/Significance This study suggest that emerging, NVT serotypes 11A and 35B deserve a close surveillance.

STAPHYLOCOCCUS AUREUS BIOFILM FORMATION ON POLYPYRROLE: AN ELECTRICAL OVERVIEW

The development of organic devices based on conducting polymers for biofilm detection requires the combination of superior electrical response and high surface area for biofilm incorporation. Polypyrrole is a potential candidate for application in biofilm detection and control due to its characteristic superior electrical response and strong interaction with bacteria, which enables the use of the bioelectric effect in resulting devices. In this study, chemically synthesized polypyrrole was applied as a support for biofilm growth of S. aureus. Modifications in the electrical response of the polymeric template were explored to identify general mechanisms established during the deposition of the biofilm.

A platform for anti-biofilm assays combining viability, biomass and matrix quantifications in susceptibility assessments of antimicrobials against Staphylococcus aureus biofilms

Bacteria can exist as planktonic, the lifestyle in which single cells exist in suspension, and as biofilms, which are surface-attached bacterial communities embedded in a selfproduced matrix. Most of the antibiotics and the methods for antimicrobial work have been developed for planktonic bacteria. However, the majority of the bacteria in natural habitats live as biofilms. Biofilms develop dauntingly fast high resistance towards conventional antibacterial treatments and thus, there is a great need to meet the demands of effective anti-biofilm therapy. In this thesis project it was attempted to fill the void of anti-biofilm screening methods by developing a platform of assays that evaluate the effect that screened compounds have on the total biomass, viability and the extracellular polysaccharide (EPS) layer of the biofilms. Additionally, a new method for studying biofilms and their interactions with compounds in a continuous flow system was developed using capillary electrochromatography (CEC). The screening platform was utilized with a screening campaign using a small library of cinchona alkaloids. The assays were optimized to be statistically robust enough for screening. The first assay, based on crystal violet staining, measures total biofilm biomass, and it was automated using a liquid handling workstation to decrease the manual workload and signal variation. The second assay, based on resazurin staining, measures viability of the biofilm, and it was thoroughly optimized for the strain used, but was then a very simple and fast method to be used for primary screening. The fluorescent resazurin probe is not toxic to the biofilms. In fact, it was also shown in this project that staining the biofilms with resazurin prior to staining with crystal violet had no effect on the latter and they can be used in sequence on the same screening plate. This sequential addition step was indeed a major improvement on the use of reagents and consumables and also shortened the work time. As a third assay in the platform a wheat germ agglutinin based assay was added to evaluate the effect a compound has on the EPS layer. Using this assay it was found that even if compounds might have clear effect on both biomass and viability, the EPS layer can be left untouched or even be increased. This is a clear implication of the importance of using several assays to be able to find “true hits” in a screening setting. In the pilot study of screening for antimicrobial and anti-biofilm effects using a cinchona alkaloid library, one compound was found to have antimicrobial effect against planktonic bacteria and prevent biofilm formation at low micromolar concentration. To eradicate biofilms, a higher concentration was needed. It was also shown that the chemical space occupied by the active compound was slightly different than the rest of the cinchona alkaloids as well as the rest of the compounds used for validatory screening during the optimization processes of the separate assays.

Activity of disinfectants and biofilm production of Corynebacterium pseudotuberculosis

To verify the occurrence of caseous lymphadenitis in sheep and goats on farms of Pernambuco, Brazil, and in animals slaughtered in two Brazilian cities (Petrolina/PE and Juazeiro/BA), and to characterize the susceptibility profile of Corynebacterium pseudotuberculosis to disinfectants and antimicrobials, and its relationship with biofilm production were the objectives of this study. 398 samples were tested for sensitivity to antimicrobial drugs, disinfectants, and biofilm production. Among the 108 samples collected on the properties, 75% were positive for C. pseudotuberculosis. Slaughterhouse samples indicated an occurrence of caseous lymphadenitis in 15.66% and 6.31% for animals slaughtered in Petrolina and Juazeiro respectively. With respect to antimicrobials, the sensitivity obtained was 100% for florfenicol and tetracycline; 99.25% for enrofloxacin, ciprofloxacin and lincomycin; 98.99% for cephalothin; 98.74% for norfloxacin and sulfazotrim; 97.74% for gentamicin; 94.22% for ampicillin; 91.71% for amoxicillin; 91.21% for penicillin G; 89.19% for neomycin and 0% for novobiocin. In analyzes with disinfectants, the efficiency for chlorhexidine was 100%, 97.20% for quaternary ammonium, 87.40% for chlorine and 84.40% for iodine. 75% of the isolates were weak or non-biofilm producers. For the consolidated biofilm, found that iodine decreased biofilm formation in 13 isolates and quaternary ammonia in 11 isolates. The reduction of the biofilm formation was observed for iodine and quaternary ammonium in consolidated biofilm formation in 33% and 28% of the isolates, respectively. The results of this study highlight the importance of establishing measures to prevent and control the disease.

NaOCl effect on biofilm produced by Staphylococcus aureus isolated from the milking environment and mastitis infected cows

Biofilms constitute a physical barrier, protecting the encased bacteria from detergents and sanitizers. The objective of this work was to analyze the effectiveness of sodium hypochlorite (NaOCl) against strains of Staphylococcus aureus isolated from raw milk of cows with subclinical mastitis and Staphylococcus aureus isolated from the milking environment (blowers and milk conducting tubes). The results revealed that, in the presence of NaOCl (150ppm), the number of adhered cells of the twelve S. aureus strains was significantly reduced. When the same strains were evaluated in biofilm condition, different results were obtained. It was found that, after a contact period of five minutes with NaOCl (150ppm), four strains (two strains from milk , one from the blowers and one from a conductive rubber) were still able to grow. Although with the increasing contact time between the bacteria and the NaOCl (150ppm), no growth was detected for any of the strains. Concerning the efficiency of NaOCl on total biofilm biomass formation by each S. aureus strain, a decrease was observed when these strains were in contact with 150 ppm NaOCl for a total period of 10 minutes. This study highlights the importance of a correct sanitation protocol of all the milk processing units which can indeed significantly reduce the presence of microorganisms, leading to a decrease of cow´s mastitis and milk contamination.

Biofilm formation by Rhodococcus equi and putative association with macrolide resistance

Abstract: Rhodococcus equi is a facultative intracellular pathogen, which cause severe pyogranulomatous pneumonia in foals and tuberculosis-like lesions in humans. Its ability to form biofilm was described in strains isolated from chronic diseases associated to treatment failures in humans. This study aimed to verify the biofilm formation by 113 R. equi isolated from equine samples (clinical and fecal) using two different methods (biofilm-culturing with and without additional glucose and epifluorescence microscopy). We also aimed to determine the efficacy of azithromycin, clarithromycin and erythromycin on R. equi in established biofilm. We found 80.5% (26/41) and 63% (58/72) biofilm-positive isolates, in fecal and clinical samples, respectively. The additional glucose increased the biofilm formation by R. equi fecal samples, but not by clinical samples. The antimicrobials tested herein were not able to eradicate R. equi in biofilm even at higher concentrations. This is the first study showing the biofilm formation by R. equi isolated from equine samples. Our findings indicate that R. equi biofilm-producers may be more resistant to the antimicrobials evaluated. Further studies are warranted to test this hypothesis.

Virulence properties of Aggregatibacter actinomycetemcomitans biofilm and characterisation of its putative cytokine exploitation

Biofilms are surface-attached multispecies microbial communities that are embedded by their self-produced extracellular polymeric substances. This lifestyle enhances the survival of the bacteria and plays a major role in many chronic bacterial infections. For instance, periodontitis is initiated by multispecies biofilms. The phases of active periodontal tissue destruction and notably increased levels of proinflammatory mediators, such as the key inflammatory mediator interleukin (IL)-1beta, are typical of the disease. The opportunistic periodontal pathogen Aggregatibacter actinomycetemcomitans is usually abundant at sites of aggressive periodontitis. Despite potent host immune system responses to subgingival invaders, A. actinomycetemcomitans is able to resist clearance attempts. Moreover, some strains of A. actinomycetemcomitans can generate genetic diversity through natural transformation, which may improve the species’ adjustment tothe subgingival environment in the long term. Some biofilm forming species are known to bind and sense human cytokines. As a response to cytokines, bacteria may increase biofilm formation and alter their expression of virulence genes. Specific outer membrane receptors for interferon-γ or IL-1β have been characterised in two Gram-negative pathogens. Because little is known about periodontal pathogens’ ability to sense cytokines, we used A. actinomycetemcomitans as a model organism to investigate how the species responds to IL-1beta. The main aims of this thesis were to explore cytokine binding on single-species A. actinomycetemcomitans biofilms and to determine the effects of cytokines on the biofilm formation and metabolic activity of the species. Additionally, the cytokine’s putative internalisation and interaction with A. actinomycetemcomitans proteins were studied. The possible impact of biofilm IL-1beta sequestering on the proliferation and apoptosis of gingival keratinocyte cells was evaluated in an organotypic mucosa co-culture model. Finally, the role of the extramembranous domain of the outer membrane protein HofQ (emHofQ) in DNA binding linked to DNA uptake in A. actinomycetemcomitans was examined. Our main finding revealed that viable A. actinomycetemcomitans biofilms can bind and take up the IL-1β produced by gingival cells. At the sites of pathogen-host interaction, the proliferation and apoptosis of gingival keratinocytes decreased slightly. Notably, the exposure of biofilms to IL-1beta caused their metabolic activity to drop, which may be linked to the observed interaction of IL-1beta with the conserved intracellular proteins DNA binding protein HU and the trimeric form of ATP synthase subunit beta. A Pasteurellaceaespecific lipoprotein, which had no previously determined function, was characterized as an IL-1beta interacting membrane protein that was expressed in the biofilm cultures of all tested A. actinomycetemcomitans strains. The use of a subcellular localisation tool combined with experimental analyses suggested that the identified lipoprotein, bacterial interleukin receptor I (BilRI), may be associated with the outer membrane with a portion of the protein oriented towards the external milieu. The results of the emHofQ study indicated that emHofQ has both the structural and functional capability to bind DNA. This result implies that emHofQ plays a role in DNA assimilation. The results from the current study also demonstrate that the Gram-negative oral species appears to sense the central proinflammatory mediator IL-1beta.

Fast and slow voltage modulation of apical Cl- permeability in toad skin at high [K+]

The influence of voltage on the conductance of toad skin was studied to identify the time course of the activation/deactivation dynamics of voltage-dependent Cl- channels located in the apical membrane of mitochondrion-rich cells in this tissue. Positive apical voltage induced an important conductance inhibition which took a few seconds to fully develop and was instantaneously released by pulse inversion to negative voltage, indicating a short-duration memory of the inhibiting factors. Sinusoidal stimulation at 23.4 mM [Cl-] showed hysteresis in the current versus voltage curves, even at very low frequency, suggesting that the rate of voltage application was also relevant for the inhibition/releasing effect to develop. We conclude that the voltage modulation of apical Cl- permeability is essentially a fast process and the apparent slow components of activation/deactivation obtained in the whole skin are a consequence of a gradual voltage build-up across the apical membrane due to voltage sharing between apical and basolateral membranes

Experimental Studies on Non-Metallic Composite Bone Implants With a Special Reference to Staphylococcal Biofilm Infection

Non-metallic implants made of bioresorbable or biostable synthetic polymers are attractive options in many surgical procedures, ranging from bioresorbable suture anchors of arthroscopic surgery to reconstructive skull implants made of biostable fiber-reinforced composites. Among other benefits, non-metallic implants produce less interference in imaging. Bioresorbable polymer implants may be true multifunctional, serving as osteoconductive scaffolds and as matrices for simultaneous delivery of bone enhancement agents. As a major advantage for loading conditions, mechanical properties of biostable fiber-reinforced composites can be matched with those of the bone. Unsolved problems of these biomaterials are related to the risk of staphylococcal biofilm infections and to the low osteoconductivity of contemporary bioresorbable composite implants. This thesis was focused on the research and development of a multifunctional implant model with enhanced osteoconductivity and low susceptibility to infection. In addition, the experimental models for assessment, diagnostics and prophylaxis of biomaterial-related infections were established. The first experiment (Study I) established an in vitro method for simultaneous evaluation of calcium phosphate and biofilm formation on bisphenol-Aglycidyldimethacrylate and triethylenglycoldimethacrylate (BisGMA-TEGDMA) thermosets with different content of bioactive glass 45S5. The second experiment (Study II) showed no significant difference in osteointegration of nanostructured and microsized polylactide-co-glycolide/β-tricalcium phosphate (PLGA /β-TCP) composites in a minipig model. The third experiment (Study III) demonstrated that positron emission tomography (PET) imaging with the novel 68Ga labelled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) CD33 related sialic-acid immunoglobulin like lectins (Siglec-9) tracer was able to detect inflammatory response to S. epidermidis and S. aureus peri-implant infections in an intraosseous polytetrafluoroethylene catheter model. In the fourth experiment (Study IV), BisGMATEGDMA thermosets coated with lactose-modified chitosan (Chitlac) and silver nanoparticles exhibited antibacterial activity against S. aureus and P. aeruginosa strains in an in vitro biofilm model and showed in vivo biocompatibility in a minipig model. In the last experiment (Study V), a selective androgen modulator (SARM) released from a poly(lactide)-co-ε-caprolactone (PLCL) polymer matrix failed to produce a dose-dependent enhancement of peri-implant osteogenesis in a bone marrow ablation model.