Antibacterial Properties of Small-Size Peptide Derived from Penetratin against Oral Streptococci

Periodontitis, an infectious disease originating from dental biofilms that causes the irreversible loss of alveolar bone, is accompanied by gradual biofilm formation and the continuous progression of inflammation. A small peptide derived from penetratin, Arg-Gln-Ile-Arg-Arg-Trp-Trp-Gln-Arg-NH₂ (RR9), appears to have antibacterial properties against selected strains associated with periodontitis. The purpose of this research is to assess the antibacterial activity and mechanism of RR9 against the initial oral colonizers Streptococci oralis, Streptococci gordonii, and Streptococci sanguinis and to investigate the cytotoxicity of RR9 on human gingival fibroblasts in vitro. The effects of RR9 on the initial oral settlers of planktonic and biofilm states were evaluated by measuring the MIC, MBC, bactericidal kinetics, and antibiofilm activity. Visual evidence and antibacterial mechanisms were identified, and the anti-inflammatory activity and cytotoxicity were measured. The results demonstrated that RR9 can inhibit the growth of streptococci in the planktonic state and during biofilm formation in vitro while keeping a low toxicity against eukaryotic cells. The antibacterial mechanism was proven to be related to the lower expression of sspA in streptococci. RR9 may be used as a potential antimicrobial and anti-infective agent for periodontal disease.     

Antibiofilm Efficacy of the Pseudomonas aeruginosa Pbunavirus vB_PaeM-SMS29 Loaded onto Dissolving Polyvinyl Alcohol Microneedles

Resistant bacteria prevail in most chronic skin wounds and other biofilm-related topical skin infections. Bacteriophages (phages) have proven their antimicrobial effectiveness for treating different antibiotic-resistant and multidrug-resistant bacterial infections, but not all phages are effective against biofilms. Phages possessing depolymerases can reach different biofilm layers; however, those that do not have depolymerase activity struggle to penetrate and navigate in the intricate 3D biofilm structure and mainly infect bacteria lodged in the outer biofilm layers. To address this, Pseudomonas aeruginosa phage vB_PaeM-SMS29, a phage with poor antibiofilm properties, was incorporated into polyvinyl alcohol (PVA, Mowiol 4:88) supplemented with 0.1% (v/v) of glycerol, and cast onto two different microneedle arrays varying in geometry. The dissolving microneedles were thoroughly characterized by microscopy, force-displacement, swelling, phage release and stability. Furthermore, 48 h-old biofilms were formed using the colony biofilm procedure (absence of broth), and the antibiofilm efficacy of the phage-loaded microneedles was evaluated by viable cell counts and microscopy and compared to free phages. The phages in microneedles were fairly stable for six months when stored at 4 °C, with minor decreases in phage titers observed. The geometry of the microneedles influenced the penetration and force-displacement characteristics but not the antimicrobial efficacy against biofilms. The two PVA microneedles loaded with phages reduced P. aeruginosa PAO1 biofilms by 2.44 to 2.76 log₁₀ CFU·cm⁻² at 24 h. These values are significantly higher than the result obtained after the treatment with the free phage (1.09 log₁₀ CFU·cm⁻²). Overall, this study shows that the distribution of phages caused by the mechanical disruption of biofilms using dissolving microneedles can be an effective delivery method against topical biofilm-related skin infections.     

Farnesol as antibiotics adjuvant in Staphylococcus epidermidis control in vitro

IntroductionFarnesol is a sesquiterpenoid that has been described as impairing bacterial growth. Therefore, the goal of this study was to compare the in vitro postantimicrobial effect (PAE) of farnesol against Staphylococcus epidermidis with the corresponding values of most common practice antibiotics and also to evaluate the combined effect of farnesol with these antibiotics against planktonic and biofilm cells.MethodsAfter exposure of S epidermidis cells to farnesol and antibiotics at minimum inhibitory concentration for 1 hour, the cells were regrown in medium without any antimicrobial agent. Cellular viability was assessed by colony-forming units, every hour for 12 hours, and then, the PAE was determined. The combined effect of farnesol (0, 30, 100 and 300 μM) with vancomycin, tetracycline and rifampicin was also evaluated, by using these antibiotics at peak serum concentration.ResultsWhen PAE is concerned, it was found that cells grown in 100 μM of farnesol behaved similarly to cells that had never been in contact with farnesol, whereas a clear difference was obtained with cells exposed to 300 μM of farnesol, displaying a longer PAE. Farnesol showed a combined effect with the tested antibiotics against planktonic cells, although this was not so evident against biofilm cells.ConclusionsDespite the reduced efficacy against biofilm cells, farnesol seems to be a potential adjuvant therapeutic agent to antibiotics against S epidermidis planktonic cells. Moreover, its long PAE makes farnesol a potential candidate in the prevention of biofilm formation because it showed to be very effective against planktonic cells alone as well.     

Isolation and Characterization of a Novel Autographiviridae Phage and Its Combined Effect with Tigecycline in Controlling Multidrug-Resistant Acinetobacter baumannii-Associated Skin and Soft Tissue Infections

Multidrug-resistant Acinetobacter baumannii (MDR A. baumannii) is one of the ESKAPE pathogens that restricts available treatment options. MDR A. baumannii is responsible for a dramatic increase in case numbers of a wide variety of infections, including skin and soft tissue infections (SSTIs), resulting in pyoderma, surgical debridement, and necrotizing fasciitis. To investigate an alternative medical treatment for SSTIs, a broad range lytic Acinetobacter phage, vB _AbP_ABWU2101 (phage vABWU2101), for lysing MDR A. baumannii in associated SSTIs was isolated and the biological aspects of this phage were investigated. Morphological characterization and genomic analysis revealed that phage vABWU2101 was a new species in the Friunavirus, Beijerinckvirinae, family Autographiviridae, and order Caudovirales. Antibiofilm activity of phage vABWU2101 demonstrated good activity against both preformed biofilms and biofilm formation. The combination of phage vABWU2101 and tigecycline showed synergistic antimicrobial activities against planktonic and biofilm cells. Scanning electron microscopy confirmed that the antibacterial efficacy of the combination of phage vABWU2101 and tigecycline was more effective than the phage or antibiotic alone. Hence, our findings could potentially be used to develop a therapeutic option for the treatment of SSTIs caused by MDR A. baumannii.     

Antimicrobial,Antibiofilm, and Antioxidant Activity of Functional Poly(Butylene Succinate) Films Modified with Curcumin and Carvacrol

The use of food industry waste as bioactive compounds in the modification of biodegradable films as food packaging remains a major challenge. This study describes the preparation and bioactivity characterization of poly(butylene succinate) (PBS)-based films with the addition of the bioactive compounds curcumin (CUR) and carvacrol (CAR). Films based on PBS modified with curcumin and carvacrol at different concentration variations (0%/0.1%/1%) were prepared by solvent casting method. The antioxidant, antimicrobial, and antibiofilm properties were investigated against bacteria (Escherichia coli, Staphylococcus aureus) and fungi (Candida albicans). As a result of the modification, the films exhibited free radicals scavenging (DPPH up to 91.47% and ABTS up to 99.21%), as well as antimicrobial (6 log, 4 log, and 2 log reductions for E. coli, S. aureus, and C. albicans, respectively, for samples modified with 1% CUR and 1% CAR) activity. Moreover, antibiofilm activity of modified materials was observed (8.22–87.91% reduction of biofilm, depending on bioactive compounds concentration). PBS films modified with curcumin and carvacrol with observed bifunctional properties have many potential applications as active packaging.     

Antibacterial and antibiofilm activities of polysaccharides,essential oil,and fatty oil extracted from Laurus nobilis growing in Lebanon

ObjectiveTo evaluate the antibacterial activity of the extracts of Laurus nobilis against three Gram-positive bacteria (Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212 and Staphylococcus epidermidis CIP 444) and two Gram-negative bacteria (Escherichia coli ATCC 35218 and Pseudomonas aeruginosa ATCC 27853). Also, the antibiofilm activity has been investigated against the biofilm produced by Staphylococcus epidermidis CIP 444.MaterialsThe polysaccharides, essential oil, and fatty oils extracted from the plant were used in broth microdilution methods to study the minimal inhibitory concentration, and then the minimal bactericidal concentration was determined.ResultsThe results showed that alginate, fucoidan, fatty oils and essential oil have good antibacterial activities against the 5 bacterial strains, and a negligible biofilm eradication activity of fucoidan, laminaran, fatty oil, and essential oil was observed, but a promising biofilm eradication activity was obtained with alginate, which showed a reduced biofilm mass even at low concentration.ConclusionsThe extracts obtained have promising antibacterial capacities which need further investigation for them to be incorporated in medical or nutritional applications.     

Bacterial Nanocellulose Fortified with Antimicrobial and Anti-Inflammatory Natural Products from Chelidonium majus Plant Cell Cultures

In this work we developed a bi-functional Bacterial-Nano-Cellulose (BNC) carrier system for cell cultures of Chelidonium majus—a medicinal plant producing antimicrobial compounds. The porous BNC was biosynthesized for 3, 5 or 7 days by the non-pathogenic Komagataeibacter xylinus bacteria and used in three forms: (1) Without removal of K. xylinus cells, (2) partially cleaned up from the remaining K. xylinus cells using water washing and (3) fully purified with NaOH leaving no bacterial cells remains. The suspended C. majus cells were inoculated on the BNC pieces in liquid medium and the functionalized BNC was harvested and subjected to scanning electron microscopy observation and analyzed for the content of C. majus metabolites as well as to antimicrobial assays and tested for potential proinflammatory irritating activity in human neutrophils. The highest content and the most complex composition of pharmacologically active substances was found in 3-day-old, unpurified BNC, which was tested for its bioactivity. The assays based on the IL-1β, IL-8 and TNF-α secretion in an in vitro model showed an anti-inflammatory effect of this particular biomatrix. Moreover, 3-day-old-BNC displayed antimicrobial and antibiofilm activity against Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. The results of the research indicated a possible application of such modified composites, against microbial pathogens, especially in local surface infections, where plant metabolite-enriched BNC may be used as the occlusive dressing.     

RNAIII-inhibiting peptide significantly reduces bacterial load and enhances the effect of antibiotics in the treatment of central venous catheter-associated Staphylococcus aureus infections

BACKGROUND: Medical devices used in clinical practice are often associated with biofilm-associated staphylococcal infections. METHODS: An in vitro antibiotic susceptibility assay of Staphylococcus aureus biofilms using 96-well polystyrene tissue-culture plates was performed to test the effects of RNAIII-inhibiting peptide (RIP), ciprofloxacin, imipenem, and vancomycin. Efficacy studies were performed using a rat model of central venous catheter (CVC)-associated infection. Twenty-four hours after implantation, the catheters were filled with RIP (1 mg/mL). Thirty minutes later, rats were challenged, via the CVC, with 1.0 x 10(6) cfu of S. aureus strain Smith diffuse. The antibiotic-lock technique was begun 24 h later. RESULTS: Minimum inhibitory concentrations of antibiotics in biofilms were at least 4-fold higher than those against the freely growing planktonic cells. When they were first treated with RIP, the cells in biofilms became as susceptible to antibiotics as did planktonic cells. These data were confirmed by the in vivo studies. In particular, when CVCs were treated with both RIP and antibiotics, the biofilm bacterial load was further reduced to 1 x 10(1) cfu/mL, and bacteremia was not detected, suggesting that there was 100% elimination of bacteremia and a 6 log10 reduction in biofilm bacterial load. CONCLUSION: RIP significantly reduces bacterial load and enhances the effect of antibiotics in the treatment of CVC-associated S. aureus infections.     

ZnO Nanoparticles-Modified Dressings to Inhibit Wound Pathogens

Zinc oxide (ZnO) nanoparticles (NPs) have been investigated for various skin therapies in recent years. These NPs can improve the healing and modulate inflammation in the wounds, but the mechanisms involved in such changes are yet to be known. In this study, we have designed a facile ZnO nano-coated dressing with improved antimicrobial efficiency against typical wound pathogens involved in biofilm and chronic infections. ZnO NPs were obtained by hydrothermal method and characterized by X-ray diffraction, scanning electron microscopy, transmission electron microscopy, and Fourier-transform infrared spectroscopy. Antibacterial and antibiofilm effects were evaluated against laboratory and clinical isolates of significant Gram-negative (Pseudomonas aeruginosa and Escherichia coli) and Gram-positive (Staphylococcus aureus and Enterococcus faecalis) opportunistic pathogens, by quantitative methods. Our results have shown that the developed dressings have a high antibacterial efficiency after 6–24 h of contact when containing 0.6 and 0.9% ZnO NPs and this effect is similar against reference and clinical isolates. Moreover, biofilm development is significantly impaired for up to three days of contact, depending on the NPs load and microbial species. These results show that ZnO-coated dressings prevent biofilm development of main wound pathogens and represent efficient candidates for developing bioactive dressings to fight chronic wounds.     

A Case of In Situ Phage Therapy against Staphylococcus aureus in a Bone Allograft Polymicrobial Biofilm Infection: Outcomes and Phage-Antibiotic Interactions

Phage therapy (PT) shows promising potential in managing biofilm infections, which include refractory orthopedic infections. We report the case of a 13-year-old girl who developed chronic polymicrobial biofilm infection of a pelvic bone allograft after Ewing’s sarcoma resection surgery. Chronic infection by Clostridium hathewayi, Proteus mirabilis and Finegoldia magna was worsened by methicillin-susceptible Staphylococcus aureus exhibiting an inducible Macrolides-Lincosamides-Streptogramin B resistance phenotype (iMLSB). After failure of conventional conservative treatment, combination of in situ anti-S. aureus PT with surgical debridement and intravenous antibiotic therapy led to marked clinical and microbiological improvement, yet failed to prevent a recurrence of infection on the midterm. This eventually led to surgical graft replacement. Multiple factors can explain this midterm failure, among which incomplete coverage of the polymicrobial infection by PT. Indeed, no phage therapy against C. hathewayi, P. mirabilis or F. magna could be administered. Phage-antibiotic interactions were investigated using OmniLog^® technology. Our results suggest that phage-antibiotic interactions should not be considered “unconditionally synergistic”, and should be assessed on a case-by-case basis. Specific pharmacodynamics of phages and antibiotics might explain these differences. More than two years after final graft replacement, the patient remains cured of her sarcoma and no further infections occurred.     

Novel Bacteriophage Specific against Staphylococcus epidermidis and with Antibiofilm Activity

Staphylococcus epidermidis has emerged as the most important pathogen in infections related to indwelling medical devices, and although these infections are not life-threatening, their frequency and the fact that they are extremely difficult to treat represent a serious burden on the public health system. Treatment is complicated by specific antibiotic resistance genes and the formation of biofilms. Hence, novel therapeutic strategies are needed to fight these infections. A novel bacteriophage CUB-EPI_14 specific to the bacterial species S. epidermidis was isolated from sewage and characterized genomically and phenotypically. Its genome contains a total of 46,098 bp and 63 predicted genes, among which some have been associated with packaging and lysis-associated proteins, structural proteins, or DNA- and metabolism-associated proteins. No lysogeny-associated proteins or known virulence proteins were identified in the phage genome. CUB-EPI_14 showed stability over a wide range of temperatures (from −20 °C to 50 °C) and pH values (pH 3–pH 12) and a narrow host range against S. epidermidis. Potent antimicrobial and antibiofilm activities were observed when the phage was tested against a highly susceptible bacterial isolate. These encouraging results open the door to new therapeutic opportunities in the fight against resilient biofilm-associated infections caused by S. epidermidis.     

Antimicrobial activity of Hibiscus sabdariffa extract against uropathogenic strains isolated from recurrent urinary tract infections

ObjectiveTo report the antimicrobial effect and biofilm forming capacity of the uropathogenic strains that have been isolated from recurrent urinary tract infections (UTIs) in the presence of Hibiscus sabdariffa (H. sabdariffa) extract.MethodsSix Escherichia coli and two Klebsiella pneumoniae isolates were collected from patients with recurrent UTIs. The susceptibility of bacterial isolates to H. sabdariffa extracts were tested by determining their minimum inhibitory concentrations (MICs), and minimum bactericidal concentration (MBC) by using the broth microdilution method in accordance to Clinical and Laboratory Standards Institute guidelines. Time-kill curves were plotted against the eight isolates based on the MIC results. The biofilm forming capacity of the isolates were evaluated using the microtiter plate assay. Detection of biofilms was done using the crystal violet staining method.ResultsVarious levels of the extracts MIC were observed against all the uropathogenic isolates. MIC values ranged from 0.5 to 4 mg/mL, and MBC values ranged from 8 to 64 mg/mL. Both the time-kill experiment and MBC-MIC ratio demonstrated that the extracts' effect was in general, bacteriostatic. The biofilm capacity inhibition assay results showed that extracts inhibited biofilm production of all the isolates. The level of biofilm inhibition however, had varied among the bacterial strains and ranged from 8%–60% reduction in optical density.ConclusionsThe results of the study support the effective potential of H. sabdariffa extract to prevent recurrent UTIs and to emphasize the significance of the plant extract, in order to approach it as a potential antimicrobial agent.     

The Effect of Liquid Rubber Addition on the Physicochemical Properties,Cytotoxicity, and Ability to Inhibit Biofilm Formation of Dental Composites

The aim of this study was to evaluate the effect of modification with liquid rubber on the adhesion to tooth tissues (enamel, dentin), wettability and ability to inhibit bacterial biofilm formation of resin-based dental composites. Two commercial composites (Flow-Art–flow type with 60% ceramic filler and Boston–packable type with 78% ceramic filler; both from Arkona Laboratorium Farmakologii Stomatologicznej, Nasutów, Poland) were modified by addition of 5% by weight (of resin) of a liquid methacrylate-terminated polybutadiene. Results showed that modification of the flow type composite significantly (p < 0.05) increased the shear bond strength values by 17% for enamel and by 33% for dentine. Addition of liquid rubber significantly (p < 0.05) reduced also hydrophilicity of the dental materials since the water contact angle was increased from 81–83° to 87–89°. Interestingly, modified packable type material showed improved antibiofilm activity against Steptococcus mutans and Streptococcus sanguinis (quantitative assay with crystal violet), but also cytotoxicity against eukaryotic cells since cell viability was reduced to 37% as proven in a direct-contact WST-8 test. Introduction of the same modification to the flow type material significantly improved its antibiofilm properties (biofilm reduction by approximately 6% compared to the unmodified material, p < 0.05) without cytotoxic effects against human fibroblasts (cell viability near 100%). Thus, modified flow type composite may be considered as a candidate to be used as restorative material since it exhibits both nontoxicity and antibiofilm properties.     

Deconstructing the Phage–Bacterial Biofilm Interaction as a Basis to Establish New Antibiofilm Strategies

The bacterial biofilm constitutes a complex environment that endows the bacterial community within with an ability to cope with biotic and abiotic stresses. Considering the interaction with bacterial viruses, these biofilms contain intrinsic defense mechanisms that protect against phage predation; these mechanisms are driven by physical, structural, and metabolic properties or governed by environment-induced mutations and bacterial diversity. In this regard, horizontal gene transfer can also be a driver of biofilm diversity and some (pro)phages can function as temporary allies in biofilm development. Conversely, as bacterial predators, phages have developed counter mechanisms to overcome the biofilm barrier. We highlight how these natural systems have previously inspired new antibiofilm design strategies, e.g., by utilizing exopolysaccharide degrading enzymes and peptidoglycan hydrolases. Next, we propose new potential approaches including phage-encoded DNases to target extracellular DNA, as well as phage-mediated inhibitors of cellular communication; these examples illustrate the relevance and importance of research aiming to elucidate novel antibiofilm mechanisms contained within the vast set of unknown ORFs from phages.     

A zinc-dependent adhesion module is responsible for intercellular adhesion in staphylococcal biofilms

Hospital-acquired bacterial infections are an increasingly important cause of morbidity and mortality worldwide. Staphylococcal species are responsible for the majority of hospital-acquired infections, which are often complicated by the ability of staphylococci to grow as biofilms. Biofilm formation by Staphylococcus epidermidis and Staphylococcus aureus requires cell-surface proteins (Aap and SasG) containing sequence repeats known as G5 domains; however, the precise role of these proteins in biofilm formation is unclear. We show here, using analytical ultracentrifugation (AUC) and circular dichroism (CD), that G5 domains from Aap are zinc (Zn²⁺)-dependent adhesion modules analogous to mammalian cadherin domains. The G5 domain dimerizes in the presence of Zn²⁺, incorporating 2–3 Zn²⁺ ions in the dimer interface. Tandem G5 domains associate in a modular fashion, suggesting a “zinc zipper” mechanism for G5 domain-based intercellular adhesion in staphylococcal biofilms. We demonstrate, using a biofilm plate assay, that Zn²⁺ chelation specifically prevents biofilm formation by S. epidermidis and methicillin-resistant S. aureus (MRSA). Furthermore, individual soluble G5 domains inhibit biofilm formation in a dose-dependent manner. Thus, the complex three-dimensional architecture of staphylococcal biofilms results from the self-association of a single type of protein domain. Surface proteins with tandem G5 domains are also found in other bacterial species, suggesting that this mechanism for intercellular adhesion in biofilms may be conserved among staphylococci and other Gram-positive bacteria. Zn²⁺ chelation represents a potential therapeutic approach for combating biofilm growth in a wide range of bacterial biofilm-related infections.     

From the Cover: Respiratory syncytial virus infection enhances Pseudomonas aeruginosa biofilm growth through dysregulation of nutritional immunity

Clinical observations link respiratory virus infection and Pseudomonas aeruginosa colonization in chronic lung disease, including cystic fibrosis (CF) and chronic obstructive pulmonary disease. The development of P. aeruginosa into highly antibiotic-resistant biofilm communities promotes airway colonization and accounts for disease progression in patients. Although clinical studies show a strong correlation between CF patients’ acquisition of chronic P. aeruginosa infections and respiratory virus infection, little is known about the mechanism by which chronic P. aeruginosa infections are initiated in the host. Using a coculture model to study the formation of bacterial biofilm formation associated with the airway epithelium, we show that respiratory viral infections and the induction of antiviral interferons promote robust secondary P. aeruginosa biofilm formation. We report that the induction of antiviral IFN signaling in response to respiratory syncytial virus (RSV) infection induces bacterial biofilm formation through a mechanism of dysregulated iron homeostasis of the airway epithelium. Moreover, increased apical release of the host iron-binding protein transferrin during RSV infection promotes P. aeruginosa biofilm development in vitro and in vivo. Thus, nutritional immunity pathways that are disrupted during respiratory viral infection create an environment that favors secondary bacterial infection and may provide previously unidentified targets to combat bacterial biofilm formation.Viral–bacterial interactions impact the development and evolution of chronic infections at many mucosal sites, including the airway (1–3). In the lung disease cystic fibrosis (CF), viral infections are linked to pulmonary function decline, antibiotic use, prolonged hospitalizations, and increased respiratory symptoms (4). Respiratory syncytial virus (RSV) is one of the most common viral copathogens in CF and is a culprit in disease progression, promoting early respiratory tract morbidity and reductions in lung function (5, 6). Moreover, beyond the morbidity associated with viral infections alone, RSV has been linked in clinical studies to the development of Pseudomonas aeruginosa coinfections and to the conversion to chronic P. aeruginosa colonization in CF patients (6–10). Although clinical associations between viral infection and the acquisition of colonizing P. aeruginosa are clear, the basic biology of this interaction is not understood.The transition of acute bacterial infections to chronic infections often involves the development of bacterial aggregates, or biofilms. The combination of an up-regulation of antibiotic resistance genes and the production of a polymeric matrix surrounding the biofilm serves to protect bacteria from the hostile environment in the host (11). The development of biofilm in human disease has been studied intensely for its involvement in disease progression in CF. Biofilm development at a mucosal surface requires initial attachment of bacteria to a surface, followed by the formation and growth of microcolonies, resulting in the development of bacterial biofilms, which can undergo regulated dispersal and ultimately seed a new surface (12, 13). Our present understanding of bacterial biofilm development is largely limited to single-organism infections. Although we have long known of polymicrobial communities colonizing human tissues, there is a surprising gap in our understanding of how these communities develop, how they impact human disease, and how host defense mechanisms influence polymicrobial infections. Because our current antimicrobial approaches have limited success for chronic infections, elucidating the mechanism by which biofilms develop during polymicrobial infections may identify new therapeutic targets to combat biofilm persistence.Many environmental cues have been described as contributing to the conversion of P. aeruginosa to a biofilm mode of growth; one such cue is iron. Nutrient iron is tightly regulated in the host through complex interactions among uptake, storage, and use in the cell. Nutritional immunity postulates that, because iron is required for microbial growth, respiration, and metabolism, the host employs many regulatory pathways to sequester free iron (14). In CF, elevated levels of iron in the airways of infected patients are correlated with frequency of exacerbation and have been proposed to play a role in airway colonization (15, 16). The sputum of CF patients contains elevated levels of ferrous iron, and these levels correlate with disease severity (17). Although increased iron in sputum is associated with CF lung disease severity, it still is unknown how iron homeostasis is altered in CF and how this alteration relates to airway infection.Using CF lung disease as a model to understand viral–bacterial interactions at a mucosal surface, we use a coculture system for bacterial biofilm development in association with the airway epithelium. Using RSV, we demonstrate that virus coinfection and the subsequent antiviral IFN response dramatically enhance the growth of P. aeruginosa biofilm in association with the airway epithelium. In addition, we show that virus infection impairs nutritional immunity, allowing the apical release of transferrin and thus increasing bioavailability of iron to promote the growth of P. aeruginosa biofilm. These findings offer new insight into the complex interaction among two pathogens and the host during polymicrobial infections and suggest a mechanism by which nutritional immunity plays a critical role in regulating pathogen persistence in the airway.     

Synthesis and Physicochemical Characterization of Novel Dicyclopropyl-Thiazole Compounds as Nontoxic and Promising Antifungals

There is a need to search for new antifungals, especially for the treatment of the invasive Candida infections, caused mainly by C. albicans. These infections are steadily increasing at an alarming rate, mostly among immunocompromised patients. The newly synthesized compounds (3a–3k) were characterized by physicochemical parameters and investigated for antimicrobial activity using the microdilution broth method to estimate minimal inhibitory concentration (MIC). Additionally, their antibiofilm activity and mode of action together with the effect on the membrane permeability in C. albicans were investigated. Biofilm biomass and its metabolic activity were quantitatively measured using crystal violet (CV) staining and tetrazolium salt (XTT) reduction assay. The cytotoxic effect on normal human lung fibroblasts and haemolytic effect were also evaluated. The results showed differential activity of the compounds against yeasts (MIC = 0.24–500 µg/mL) and bacteria (MIC = 125–1000 µg/mL). Most compounds possessed strong antifungal activity (MIC = 0.24–7.81 µg/mL). The compounds 3b, 3c and 3e, showed no inhibitory (at 1/2 × MIC) and eradication (at 8 × MIC) effect on C. albicans biofilm. Only slight decrease in the biofilm metabolic activity was observed for compound 3b. Moreover, the studied compounds increased the permeability of the membrane/cell wall of C. albicans and their mode of action may be related to action within the fungal cell wall structure and/or within the cell membrane. It is worth noting that the compounds had no cytotoxicity effect on pulmonary fibroblasts and erythrocytes at concentrations showing anticandidal activity. The present studies in vitro confirm that these derivatives appear to be a very promising group of antifungals for further preclinical studies.     

Effect of Surface Tooling Techniques of Medical Titanium Implants on Bacterial Biofilm Formation In Vitro

The aim of this study was to assess the biofilm formation of Streptococcus mutans, Staphylococcus aureus, Enterococcus faecalis, and Escherichia coli on titanium implants with CAD-CAM tooling techniques. Twenty specimens of titanium were studied: Titanium grade 2 tooled with a Planmeca CAD-CAM milling device (TiGrade 2), Ti₆Al₄V grade 5 as it comes from CAD-DMLS device (computer aided design-direct metal laser sintering device) (TiGrade 5), Ti₆Al₄V grade 23 as it comes from a CAD-CAM milling device (TiGrade 23), and CAD-DMLS TiGrade 5 polished with an abrasive disc (TiGrade 5 polished). Bacterial adhesion on the implants was completed with and without saliva treatment to mimic both extraoral and intraoral surgical methods of implant placement. Five specimens/implant types were used in the bacterial adhesion experiments. Autoclaved implant specimens were placed in petri plates and immersed in saliva solution for 30 min at room temperature and then washed 3× with 1× PBS. Bacterial suspensions of each strain were made and added to the specimens after saliva treatment. Biofilm was allowed to form for 24 h at 37 °C and the adhered bacteria was calculated. Tooling techniques had an insignificant effect on the bacterial adhesion by all the bacterial strains studied. However, there was a significant difference in biofilm formation between the saliva-treated and non-saliva-treated implants. Saliva contamination enhanced S. mutans, S. aureus, and E. faecalis adhesion in all material types studied. S. aureus was found to be the most adherent strain in the saliva-treated group, whereas E. coli was the most adherent strain in the non-saliva-treated group. In conclusion, CAD-CAM tooling techniques have little effect on bacterial adhesion. Saliva coating enhances the biofilm formation; therefore, saliva contamination of the implant must be minimized during implant placement. Further extensive studies are needed to evaluate the effects of surface treatments of the titanium implant on soft tissue response and to prevent the factors causing implant infection and failure.     

In vitro and in vivo activity of EDTA and antibacterial agents against the biofilm of mucoid <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Purpose

Refractory infection caused by bacterial biofilm is an important clinical problem. Pseudomonas aeruginosa is a common pathogen responsible for persistent and chronic biofilm infections. We aimed to explore the in vitro and in vivo activity of ethylenediamine tetraacetic acid (EDTA) in combination with antibacterial agents against mucoid P. aeruginosa biofilm.

Methods

The minimal inhibitory concentration (MIC) and minimal bactericidal concentration of ciprofloxacin, gentamicin, and ampicillin alone or with EDTA against P. aeruginosa were determined in vitro. Extracellular polysaccharides (EPS) and structural parameters of the biofilm were monitored. P. aeruginosa was aerosolized and delivered into the lungs of guinea pigs, which were treated with ciprofloxacin with or without EDTA. The colony-forming units (CFUs) of P. aeruginosa were determined from the lungs.

Results

EDTA reduced the MIC of ciprofloxacin and ampicillin by about 30-fold and that of gentamicin by twofold. EDTA reduced the biofilm EPS and the proportion of viable bacteria. The thickness, average diffusion distance, and textural entropy of EDTA-treated biofilm were significantly decreased. EDTA plus antibiotics reduced the colony counting from 10⁷ to 10³ CFU/mL. In vivo, EDTA plus ciprofloxacin had a significantly lower mean CFU/g of lung tissue (EDTA + ciprofloxacin 1.3 ± 0.19; EDTA 4.4 ± 0.57; ciprofloxacin 4.2 ± 0.47), and lung lesions were less severe compared with the single treatment groups.

Conclusions

EDTA can destroy the biofilm structures of mucoid P. aeruginosa in vitro. Moreover, EDTA and ciprofloxacin had a significant bactericidal effect against biofilm in vivo.

     

Real-time mapping of a hydrogen peroxide concentration profile across a polymicrobial bacterial biofilm using scanning electrochemical microscopy

Quantitative detection of hydrogen peroxide in solution above a Streptococcus gordonii (Sg) bacterial biofilm was studied in real time by scanning electrochemical microscopy (SECM). The concentration of hydrogen peroxide was determined to be 0.7 mM to 1.6 mM in the presence of 10 mM glucose over a period of 2 to 8 h. The hydrogen peroxide production measured was higher near the biofilm surface in comparison to Sg grown planktonically. Differential hydrogen peroxide production was observed both by fluorometric as well as by SECM measurements. The interaction between two different species in a bacterial biofilm of Sg and Aggregatibacter actinomycetemcomitans (Aa) in terms of hydrogen peroxide production was also studied by SECM. One-directional y-scan SECM measurements showed the unique spatial mapping of hydrogen peroxide concentration across a mixed species biofilm and revealed that hydrogen peroxide concentration varies greatly dependent upon local species composition.