The Development and Characterisation of a P(3HB-co-4HB)-Bioactive Glass-Graphene Hydrogel as a Potential Formulation for Biomedical and Therapeutical Translation.

The clinical management of wounds is known to be a significant challenge: not only does the dressing need to ensure and provide the appropriate barrier and healing characteristics, but consideration of patient compliance concerning comfort, functionality, and practicality also needs to be included. The poly(3-hydroxybutyrate-co-4-hydroxubutyrate) (P(3HB-co-4HB)) copolymer, isolated from Cupriavidus malaysiensis USM1020 (C. malaysiensis USM1020), was produced in the presence of excess carbon sources (1,4-butanediol and 1,6-hexanediol) using either a shake flask cultivation process or a bioreactor fermentation system. P(3HB-co-4HB) is widely known to be biodegradable and highly biocompatible and contains a tuneable 4HB monomer molar fraction, which is known to affect the final physicochemical properties of the intracellular copolymer. In this paper, we describe not only the fabrication of the polymeric gel but also its optimised profiling using a range of physical and mechanical techniques, i.e., SEM, FTIR, DMA, DSC, and WCA. The further enhancement of the gel through additional functionalisation with sol-gel-derived bioactive glass and liquid-exfoliated graphene was also investigated. The biocompatibility and biological characterisation of the substrates was assessed using murine osteoblasts (MC3T3), human primary dermal fibroblasts (HDFs), human fibroblast (BJ) cells, and standard cell culture assays (i.e., metabolic activity, LDH release, and live/dead staining). In short, P(3HB-co-4HB) was successfully isolated from the bacteria, with the defined physico-chemical profiles dependent on the culture substrate and culturing platform used. The additional enhancement of the copolymer with bioactive glass and/or graphene was also demonstrated by varying the combination loading of the materials, i.e., graphene resulted in an increase in tensile strength (~11 MPa) and the wettability increased following the incorporation of bioactive glass and 0.01 wt% graphene (WCA ~46.3°). No detrimental effects in terms of biocompatibility were noticed during the 7 days of culture in the primary and established cell lines. This study demonstrates the importance of optimising each of the individual components within the biocomposite and their relationship concerning the fine-tuning of the material's properties, thus targeting and impacting the endpoint application.

Zn and N Codoped TiO2 Thin Films: Photocatalytic and Bactericidal Activity.

We explore a series of Zn and N codoped TiO2 thin films grown using chemical vapor deposition. Films were prepared with various concentrations of Zn (0.4-2.9 at. % Zn vs Ti), and their impact on superoxide formation, photocatalytic activity, and bactericidal properties were determined. Superoxide (O2•-) formation was assessed using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium sodium salt (XTT) as an indicator, photocatalytic activity was determined from the degradation of stearic acid under UVA light, and bactericidal activity was assessed using a Gram-negative bacterium E. coli under both UVA and fluorescent light (similar to what is found in a clinical environment). The 0.4% Zn,N:TiO2 thin film demonstrated the highest formal quantum efficiency in degrading stearic acid (3.3 × 10-5 molecules·photon-1), while the 1.0% Zn,N:TiO2 film showed the highest bactericidal activity under both UVA and fluorescent light conditions (>3 log kill). The enhanced efficiency of the films was correlated with increased charge carrier lifetime, supported by transient absorption spectroscopy (TAS) measurements.

Crystal Violet-Impregnated Slippery Surface to Prevent Bacterial Contamination of Surfaces.

Biofilms which are self-organized communities can contaminate various infrastructural systems. Preventing bacterial adhesion on surfaces is more desirable than cleaning or disinfection of bacteria-contaminated surfaces. In this study, a 24 h bacterial adhesion test showed that "slippery surfaces" had increased resistance to bacterial contamination compared to polydimethylsiloxane and superhydrophobic surfaces. However, it did not completely inhibit bacterial attachment, indicating that it only retards surface contamination by bacteria. Hence, a strategy of killing bacteria with minimal bacterial adhesion was developed. A crystal violet-impregnated slippery (CVIS) surface with bactericidal and slippery features was produced through a simple dipping process. The CVIS surface had a very smooth and lubricated surface that was highly repellent to water and blood contamination. Bactericidal tests against Escherichia coli and Staphylococcus aureus showed that the CVIS surface exhibited bactericidal activity in dark and also showed significantly enhanced bactericidal activity (>3 log reduction in bacteria number) in white light.

Enhanced Photocatalytic and Antibacterial Ability of Cu-Doped Anatase TiO2 Thin Films: Theory and Experiment.

Multifunctional thin films which can display both photocatalytic and antibacterial activity are of great interest industrially. Here, for the first time, we have used aerosol-assisted chemical vapor deposition to deposit highly photoactive thin films of Cu-doped anatase TiO2 on glass substrates. The films displayed much enhanced photocatalytic activity relative to pure anatase and showed excellent antibacterial (vs Staphylococcus aureus and Escherichia coli) ability. Using a combination of transient absorption spectroscopy, photoluminescence measurements, and hybrid density functional theory calculations, we have gained nanoscopic insights into the improved properties of the Cu-doped TiO2 films. Our analysis has highlighted that the interactions between substitutional and interstitial Cu in the anatase lattice can explain the extended exciton lifetimes observed in the doped samples and the enhanced UV photoactivities observed.

Adhesion of Methicillin-Resistant Staphylococcus aureus and Candida albicans to Parylene-C-Coated Polymethyl Methacrylate.

PURPOSE: To determine whether coating polymethyl methacrylate (PMMA) discs with Parylene-C would reduce Staphylococcus aureus and Candida albicans biofilm formation. MATERIALS AND METHODS: MRSA and Candida albicans single and dual biofilms were grown for 48 hours in artificial saliva on parylene-C-coated or uncoated PMMA, and the viable biofilm colony-forming units were counted. RESULTS: There was no significant difference in the count of viable methicillin-resistant Staphylococcus aureus or Candida albicans recovered from single- or dual-species biofilms between coated and uncoated PMMA discs. CONCLUSION: Parylene-C does not prevent biofilm formation on PMMA.

Photobactericidal Activity of Dual Dyes Encapsulated in Silicone Enhanced by Silver Nanoparticles.

Crystal violet (CV) and methylene blue (MB) dyes with silver (Ag) nanoparticles (NPs) were encapsulated into silicone to produce light-activated antimicrobial surfaces. Optical microscopy and X-ray photoelectron spectroscopy showed that CV and MB were diffused throughout the silicone samples and that Ag NPs were successfully encapsulated by the swell-encapsulation-shrink process. Antimicrobial tests on Staphylococcus aureus and Escherichia coli showed that CV/MB-encapsulated silicone samples have stronger photobactericidal activity than CV or MB samples and the addition of Ag NPs significantly enhanced the antimicrobial activity under white light. The number of viable bacteria decreased below the detection limit (below <103 CFU) on the silicone-incorporating CV/MB/Ag NPs within 3 h for S. aureus and within 5 h for E. coli. In leaching tests over 216 h, the amount of dye leaching from the samples was barely detectable (<0.02 ppm). These surfaces have a potential for use in healthcare settings to decrease hospital-associated infections.

The Anti-Biofouling Properties of Superhydrophobic Surfaces are Short-Lived.

Superhydrophobic surfaces are present in nature on the leaves of many plant species. Water rolls on these surfaces, and the rolling motion picks up particles including bacteria and viruses. Man-made superhydrophobic surfaces have been made in an effort to reduce biofouling. We show here that the anti-biofouling property of a superhydrophobic surface is due to an entrapped air-bubble layer that reduces contact between the bacteria and the surface. Further, we showed that prolonged immersion of superhydrophobic surfaces in water led to loss of the bubble-layer and subsequent bacterial adhesion that unexpectedly exceeded that of the control materials. This behavior was not restricted to one particular type of material but was evident on different types of superhydrophobic surfaces. This work is important in that it suggests that superhydrophobic surfaces may actually encourage bacterial adhesion during longer term exposure.

Bicarbonate induces high-level resistance to the human antimicrobial peptide LL-37 in Staphylococcus aureus small colony variants.

Objectives: Staphylococcus aureus small colony variants (SCVs) cause persistent infections and are resistant to cationic antibiotics. Antimicrobial peptides (AMPs) have been suggested as promising alternatives for treating antibiotic-resistant bacteria. We investigated the capacity of the human cationic AMP LL-37 to kill SCVs in the presence of physiological concentrations of bicarbonate, which are reported to alter bacterial membrane permeability and change resistance of bacteria to AMPs. Methods: MBCs of LL-37 for S. aureus SCVs with mutations in different genes in the presence and absence of bicarbonate were determined. Results: In the absence of bicarbonate, SCVs of S. aureus strains LS-1 and 8325-4 had the same level of resistance to LL-37 as the parental strain (8 mg/L). In the presence of bicarbonate, hemB, menD and aroD SCVs of LS-1 had high-level resistance to LL-37 (≥128 mg/L) compared with the parental strain (16 mg/L). However, only the aroD SCV of strain 8324-5 showed high-level resistance. 8325-4 harbours mutations in two genes, tcaR and rsbU, which are involved in antimicrobial sensing and the stress response, respectively. When rsbU was repaired in 8325-4 it displayed high-level resistance to LL-37 in the presence of bicarbonate. This phenotype was lost when tcaR was also repaired, demonstrating that RsbU and TcaR are involved in LL-37 resistance in the presence of bicarbonate. Conclusions: S. aureus SCVs would be resistant to high concentrations of LL-37 in niches where there are physiological concentrations of bicarbonate and therefore this AMP may not be effective in combating SCVs.

Superhydrophobic and White Light-Activated Bactericidal Surface through a Simple Coating.

Bacterial adhesion and proliferation on surfaces are a challenge in medical and industrial fields. Here, a simple one-step technique is reported to fabricate self-cleaning and bactericidal surfaces. White, blue, and violet paints were produced using titanium dioxide nanoparticles, 1H,1H,2H,2H-perfluorooctyltriethoxysilane, crystal violet, toluidine Blue O, and ethanol solution. All of the painted surfaces showed superhydrophobicity in air, and even after hexadecane oil contamination, they retained water repellency and self-cleaning properties. In an assay of bacterial adhesion, significant reductions (>99.8%) in the number of adherent bacteria were observed for all the painted surfaces. In bactericidal tests, the painted surfaces not only demonstrated bactericidal activity against Staphylococcus aureus and Escherichia coli in the dark but also induced very potent photosensitization (>4.4 log reduction in the number of viable bacteria on the violet painted surface) under white light illumination. The technique that we developed here is general and can be used on a wide range of substrates such as paper, glass, polymers, and others.

Aggregatibacter actinomycetemcomitans serotype prevalence and antibiotic resistance in a UK population with periodontitis.

OBJECTIVES: Aggregatibacter actinomycetemcomitans is a recognised pathogen involved in aggressive periodontitis. Seven serotypes of A. actinomycetemcomitans exist with a range of virulence and distribution dependent on ethnicity and geography. The ability of A. actinomycetemcomitans to invade soft tissue can necessitate the use of systemic antibiotics for treatment, however variations in its antibiotic susceptibility exist dependent on geographical location. METHODS: Serotypes of A. actinomycetemcomitans isolates from a UK cohort of 50 patients with aggressive periodontitis were determined by PCR. Resistance of the isolates to eight antibiotics [penicillin (1U), amoxicillin (2µg), amoxicillin/clavulanic acid (30µg), metronidazole (5µg), clindamycin (2µg), tetracycline (10µg), ciprofloxacin (5µg) and ceftazidime (30µg)] were determined by disk diffusion according to BSAC guidelines. RESULTS: Prevalences of serotypes a, c, b, e and mixed serotypes were 48%, 22%, 2%, 2% and 12%, respectively. The serotype of isolates from seven patients (14%) could not be deduced by PCR. Of the 56 isolates tested, 100% were resistant to penicillin and metronidazole, 87.5% to clindamycin, 83.9% to amoxicillin and 76.8% to ceftazidime. Low rates of resistance to tetracycline (8.9% resistant) and amoxicillin/clavulanic acid (14.3% resistant) were observed, whereas no isolates were resistant to ciprofloxacin. CONCLUSIONS: As in a number of publications the suggested treatment of aggressive periodontitis includes the combined use of amoxicillin with metronidazole, these results highlight the need for culture and antimicrobial susceptibility investigations in patients with aggressive periodontitis prior to systemic use of antibiotics concomitantly to periodontal therapy.

An aroD Ochre Mutation Results in a Staphylococcus aureus Small Colony Variant That Can Undergo Phenotypic Switching via Two Alternative Mechanisms.

Staphylococcus aureus can undergo phenotypic switching between a normal colony phenotype (NCP) and a small colony variant (SCV). The SCV phenotype confers increased antibiotic resistance and the capacity to persist within human tissues and cells, and because these cells can revert back to the NCP they cause chronic and/or recurrent infections that are very difficult to treat. A complete picture of the genetic events that can lead to phenotypic switching in S. aureus is currently lacking. We describe the selection of an SCV with a previously unreported genetic alteration leading to an ochre mutation of aroD. In addition to the known mechanisms of phenotypic switching between the SCV and the NCP we describe a previously unreported mechanism involving tRNA ochre suppressors arising. The ochre suppressor strains had wild-type growth rates and restored antibiotic sensitivity, similar to the wild-type strain. However, whilst they had increased virulence compared to the SCV parent strain, their virulence was not restored to that of the NCP parental strain. These findings establish that phenotypic switching between the NCP and SCV states can give rise to strains with different pathogenic potential.

Antimicrobial Properties of Copper-Doped ZnO Coatings under Darkness and White Light Illumination.

We report the first antimicrobial study of transparent and robust Cu-doped ZnO coatings that displayed potent antimicrobial activity that resulted in bacterial (Escherichia coli) reduction below detection limits within 6 h of illumination via a white light source that is found in hospital environments. The same bacterial reduction rate was observed even under darkness for 4.0 atom % Cu-doped ZnO films thus providing an efficient 24 h disinfection. All films were produced via a novel, inexpensive, and easily scalable route and were also thoroughly analyzed for their material properties.

Light activated antimicrobial agents can inactivate oral malodour causing bacteria.

Oral malodour is a common condition which affects a large proportion of the population, resulting in social, emotional and psychological stress. Certain oral bacteria form a coating called a biofilm on the tongue dorsum and degrade organic compounds releasing volatile sulfur compounds that are malodourous. Current chemical treatments for oral malodour such as mouthwashes containing chlorhexidine or essential oils, are not sufficiently effective at reducing the bacterial load on the tongue. One potential alternative to current chemical treatments for oral malodour is the use of light activated antimicrobial agents (LAAAs), which display no toxicity or antimicrobial activity in the dark, but when exposed to light of a specific wavelength produce reactive oxygen species which induce damage to target cells in a process known as photodynamic inactivation. This study aimed to determine whether oral malodour causing bacteria were susceptible to lethal photosensitization. Five bacterial species that are causative agents of oral malodour were highly sensitive to lethal photosensitization and were efficiently killed by methylene blue in conjunction with 665 nm laser light. Between 4.5-5 log10 reductions in the number of viable bacteria were achieved with 20 µM methylene blue and 14.53 J cm-2 laser light for Porphyromonas gingivalis, Prevotella intermedia, Peptostreptococcus anaerobius and Solobacterium moorei. The number of viable cells fell below the limit of detection in the case of Fusobacterium nucleatum. These findings demonstrate that methylene blue in combination with 665 nm laser light is effective at killing bacteria associated with oral malodour, suggesting photodynamic therapy could be a viable treatment option for oral malodour.

Photodynamic inactivation of Candida albicans by hematoporphyrin monomethyl ether.

AIM: To evaluate the capacity of hematoporphyrin monomethyl ether (HMME) in the presence of light to cause photodynamic inactivation (PDI) of Candida albicans. MATERIALS & METHODS: HMME photoactivity was evaluated against azole-susceptible and -resistant C. albicans. The mechanisms by which PDI of C. albicans occurred were also investigated. RESULTS: HMME-mediated PACT caused a dose-dependent inactivation of azole-susceptible and -resistant C. albicans. Incubation with 10 µM HMME and irradiation with 72 J cm(-2) light decreased the viability of C. albicans by 7 log10, induced damage of genomic DNA, led to loss of cellular proteins and damaged the cell wall, membrane and intracellular targets. CONCLUSION: Candida albicans can be effectively inactivated by HMME in the presence of light, and HMME-mediated PACT shows its potential as an antifungal treatment.

Photodynamic inactivation of Klebsiella pneumoniae biofilms and planktonic cells by 5-aminolevulinic acid and 5-aminolevulinic acid methyl ester.

The treatment of Klebsiella pneumoniae, particularly extended-spectrum ß-lactamase (ESBL)-producing K. pneumoniae, is currently a great challenge. Photodynamic antimicrobial chemotherapy is a promising approach for killing antibiotic-resistant bacteria. The aim of this study was to evaluate the capacity of 5-aminolevulinic acid (5-ALA) and its derivative 5-ALA methyl ester (MAL) in the presence of white light to cause photodynamic inactivation (PDI) of K. pneumoniae planktonic and biofilm cells. In the presence of white light, 5-ALA and MAL inactivated planktonic cells in a concentration-dependent manner. Biofilms were also sensitive to 5-ALA and MAL-mediated PDI. The mechanisms by which 5-ALA and MAL caused PDI of ESBL-producing K. pneumonia were also investigated. Exposure of K. pneumonia to light in the presence of either 5-ALA or MAL induced cleavage of genomic DNA and the rapid release of intracellular biopolymers. Intensely denatured cytoplasmic contents and aggregated ribosomes were also detected by transmission electron microscopy. Scanning electron microscopy showed that PDI of biofilms caused aggregated bacteria to detach and that the bacterial cell envelope was damaged. This study provides insights into 5-ALA and MAL-mediated PDI of ESBL-producing K. pneumoniae.

Antibacterial properties of Cu-ZrO2 thin films prepared via aerosol assisted chemical vapour deposition.

The antibacterial properties of a Cu-ZrO2 film grown via aerosol assisted chemical vapour deposition are presented. The composite film showed high activity against E. coli (Gram-negative) and S. aureus (Gram-positive) bacteria with 5 log10 (E. coli) and 4 log10 (S. aureus) decrease in viable bacteria achieved within 20 and 60 minutes respectively. These results were comparable to a pure copper film that was prepared under the same conditions. The composite film was characterized for material properties using a range of techniques including X-ray photoemission and X-ray diffraction.

Oxacillin sensitization of methicillin-resistant Staphylococcus aureus and methicillin-resistant Staphylococcus pseudintermedius by antisense peptide nucleic acids in vitro.

BACKGROUND: Antibiotic resistance genes can be targeted by antisense agents, which can reduce their expression and thus restore cellular susceptibility to existing antibiotics. Antisense inhibitors can be gene and pathogen specific, or designed to inhibit a group of bacteria having conserved sequences within resistance genes. Here, we aimed to develop antisense peptide nucleic acids (PNAs) that could be used to effectively restore susceptibility to ß-lactams in methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP). RESULTS: Antisense PNAs specific for conserved regions of the mobilisable gene mecA, and the growth essential gene, ftsZ, were designed. Clinical MRSA and MRSP strains of high oxacillin resistance were treated with PNAs and assayed for reduction in colony forming units on oxacillin plates, reduction in target gene mRNA levels, and cell size. Anti-mecA PNA at 7.5 and 2.5 µM reduced mecA mRNA in MRSA and MRSP (p < 0.05). At these PNA concentrations, 66 % of MRSA and 92 % of MRSP cells were killed by oxacillin (p < 0.01). Anti-ftsZ PNA at 7.5 and 2.5 µM reduced ftsZ mRNA in MRSA and MRSP, respectively (p ≤ 0.05). At these PNA concentrations, 86 % of MRSA cells and 95 % of MRSP cells were killed by oxacillin (p < 0.05). Anti-ftsZ PNAs resulted in swelling of bacterial cells. Scrambled PNA controls did not affect MRSA but sensitized MRSP moderately to oxacillin without affecting mRNA levels. CONCLUSIONS: The antisense PNAs effects observed provide in vitro proof of concept that this approach can be used to reverse ß-lactam resistance in staphylococci. Further studies are warranted as clinical treatment alternatives are needed.

Pancreatic amylase is an environmental signal for regulation of biofilm formation and host interaction in Campylobacter jejuni.

Campylobacter jejuni is a commensal bacterium in the intestines of animals and birds and a major cause of food-borne gastroenteritis in humans worldwide. Here we show that exposure to pancreatic amylase leads to secretion of an α-dextran by C. jejuni and that a secreted protease, Cj0511, is required. Exposure of C. jejuni to pancreatic amylase promotes biofilm formation in vitro, increases interaction with human epithelial cell lines, increases virulence in the Galleria mellonella infection model, and promotes colonization of the chicken ileum. We also show that exposure to pancreatic amylase protects C. jejuni from stress conditions in vitro, suggesting that the induced α-dextran may be important during transmission between hosts. This is the first evidence that pancreatic amylase functions as an interkingdom signal in an enteric microorganism.

Identification of an antibacterial protein by functional screening of a human oral metagenomic library.

Screening of a bacterial artificial chromosome (BAC) library containing metagenomic DNA from human plaque and saliva allowed the isolation of four clones producing antimicrobial activity. Three of these were pigmented and encoded homologues of glutamyl-tRNA reductase (GluTR), an enzyme involved in the C5 pathway leading to tetrapyrole synthesis, and one clone had antibacterial activity with no pigmentation. The latter contained a BAC with an insert of 15.6 kb. Initial attempts to localize the gene(s) responsible for antimicrobial activity by subcloning into pUC-based vectors failed. A new plasmid for toxic gene expression (pTGEX) was designed enabling localization of the antibacterial activity to a 4.7-kb HindIII fragment. Transposon mutagenesis localized the gene to an open reading frame of 483 bp designated antibacterial protein1 (abp1). Abp1 was 94% identical to a hypothetical protein of Neisseria subflava (accession number WP_004519448.1). An Escherichia coli clone expressing Abp1 exhibited antibacterial activity against Bacillus subtilis BS78H, Staphylococcus epidermidis NCTC 11964 and B4268, and S. aureus NCTC 12493,ATCC 35696 and NCTC 11561. However, no antibacterial activity was observed against Pseudomonas aeruginosa ATCC 9027, N. subflava ATCC A1078, E. coli K12 JM109 and BL21(DE3) Fusobacterium nucleatum ATCC 25586 and NCTC 11326, Prevotella intermedia ATCC 25611, Veillonella parvula ATCC 10790 or Lactobacillus casei NCTC 6375.