Ching-Tsan Huang

Quorum sensing in Pseudomonas aeruginosa controls expression of catalase and superoxide dismutase genes and mediates biofilm susceptibility to hydrogen peroxide.

Quorum sensing (QS) governs the production of virulence factors and the architecture and sodium dodecyl sulphate (SDS) resistance of biofilm‐grown Pseudomonas aeruginosa. P. aeruginosa QS requires two transcriptional activator proteins known as LasR and RhlR and their cognate autoinducers PAI‐1 (N‐(3‐oxododecanoyl)‐l‐homoserine lactone) and PAI‐2 (N‐butyryl‐l‐homoserine lactone) respectively. This study provides evidence of QS control of genes essential for relieving oxidative stress. Mutants devoid of one or both autoinducers were more sensitive to hydrogen peroxide and phenazine methosulphate, and some PAI mutant strains also demonstrated decreased expression of two superoxide dismutases (SODs), Mn‐SOD and Fe‐SOD, and the major catalase, KatA. The expression of sodA (encoding Mn‐SOD) was particularly dependent on PAI‐1, whereas the influence of autoinducers on Fe‐SOD and KatA levels was also apparent but not to the degree observed with Mn‐SOD. β‐Galactosidase reporter fusion results were in agreement with these findings. Also, the addition of both PAIs to suspensions of the PAI‐1/2‐deficient double mutant partially restored KatA activity, while the addition of PAI‐1 only was sufficient for full restoration of Mn‐SOD activity. In biofilm studies, catalase activity in wild‐type bacteria was significantly reduced relative to planktonic bacteria; catalase activity in the PAI mutants was reduced even further and consistent with relative differences observed between each strain grown planktonically. While wild‐type and mutant biofilms contained less catalase activity, they were more resistant to hydrogen peroxide treatment than their respective planktonic counterparts. Also, while catalase was implicated as an important factor in biofilm resistance to hydrogen peroxide insult, other unknown factors seemed potentially important, as PAI mutant biofilm sensitivity appeared not to be incrementally correlated to catalase levels.

Spatial variations in growth rate within Klebsiella pneumoniae colonies and biofilm.

The use of acridine orange to visualize and quantify spatial variations in growth rate within Klebsiella pneumoniae colonies and biofilm was investigated. Bacterial colonies supported on polycarbonate filter membranes were grown on R2A agar plates. Some colonies were sampled for cell enumeration, while others were cryoembedded, sectioned, and stained with the fluorescent nucleic acid stain acridine orange. Spatial patterns of fluorescent color and intensity with depth in the colony were quantified using confocal microscopy and image analysis of stained cross sections. Colonies sampled in the midexponential phase were thin (20 μm), had high average specific growth rates (>1 h−1), and had all the cells stained bright orange. Colonies sampled after more than 24 h of growth were thick (>200 μm) and were growing slowly (μ < 0.15 h−1). These older colonies were characterized by distinct bands of orange at the colony edges and a dark green center. Stained biofilm cross sections displayed a similar orange band at the biofilm−bulk fluid interface and a green interior. Colony‐average specific growth rates, determined by calculating the local slope of the cell accumulation versus time data, were correlated with colony‐average fluorescence intensities. There was no correlation betweeen average specific growth rate and orange or green intensity individually, but growth rate did correlate with the orange:green intensity ratio (r2 = 0.57). The resulting regression was used to predict specific growth rate profiles within colonies. These profiles indicated that bacteria were growing rapidly near the air and agar interfaces and more slowly in the center of the colonies when thicker than about 30 μm. The dimension of the orange bands ranged from 10 to 30 μm, which may indicate the thickness of growing regions. The inherent variability associated with this technique suggests that it is best applied in single species systems and that the results should be regarded as qualitative in nature.

Spatial Distribution and Coexistence of Klebsiella pneumoniae and Pseudomonas aeruginosa in Biofilms

A bstractThe heterotrophic bacteria Klebsiella pneumoniae and Pseudomonas aeruginosa stably coexisted in laboratory-grown biofilms, even though the growth rate of K. pneumoniae was twice that of P. aeruginosa under planktonic growth conditions. The failure of K. pneumoniae to displace P. aeruginosa from the biofilm could not be attributed to concentration gradients of the limiting nutrient (glucose) arising from the interaction of reaction and diffusion. Comparisons of the growth rates of the two species in mono- and binary-population biofilms suggested partial segregation of the two species in the latter. We used a fluorescently labeled monoclonal antibody to examine the spatial distribution of K. pneumoniae in frozen cross sections of biofilm to confirm this segregation. K. pneumoniae microcolonies resided on top of, or intermixed with, a base film of P. aeruginosa. We hypothesize that microscale structural heterogeneity and differing rates of bacterial attachment and detachment of the two species are responsible for coexistence in this system.

An immunomodulatory protein, Ling Zhi-8, induced activation and maturation of human monocyte-derived dendritic cells by the NF-κB and MAPK pathways

Ganoderma lucidum, an oriental medicinal mushroom, has been widely used in Asia to promote health and longevity. LZ‐8 is a protein derived from the fungus G. lucidum and has immunomodulatory capacities. In this study, we investigated the immune modulatory effects of rLZ‐8 on human monocyte‐derived DCs. Treatment of DC with rLZ‐8 resulted in the enhanced cell‐surface expression of CD80, CD86, CD83, and HLA‐DR, as well as the enhanced production of IL‐12 p40, IL‐10, and IL‐23, and the capacity for endocytosis was suppressed in DCs. In addition, treatment of DCs with rLZ‐8 resulted in an enhanced, naïve T cell‐stimulatory capacity and increased, naïve T cell secretion of IFN‐γ and IL‐10. Neutralization with antibodies against TLR4 inhibited the rLZ‐8‐induced production of IL‐12 p40 and IL‐10 in DCs. rLZ‐8 can stimulate TLR4 or TLR4/MD2‐transfected HEK293 cells to produce IL‐8. These results suggested an important role for TLR4 in signaling DCs upon incubation with rLZ‐8. Further study showed that rLZ‐8 was able to augment IKK, NF‐κB activity, and also IκBα and MAPK phosphorylation. Further, inhibition of NF‐κB by helenalin prevented the effects of rLZ‐8 in the expression of CD80, CD86, CD83, and HLA‐DR and production of IL‐12 p40 and IL‐10 in various degrees. To confirm the in vitro data, we investigated the effect of rLZ‐8 further on antigen‐specific antibody and cytokine production in BALB/c mice. Immunization with OVA/rLZ‐8 showed that the anti‐OVA IgG2a, IFN‐γ, and IL‐2 were increased significantly compared with OVA alone in BALB/c mice. In conclusion, our experiments demonstrated that rLZ‐8 can effectively promote the activation and maturation of immature DCs, preferring a Th1 response, suggesting that rLZ‐8 may possess a potential effect in regulating immune responses.

Chitosan Augments Photodynamic Inactivation of Gram-Positive and Gram-Negative Bacteria

ABSTRACT Antimicrobial photodynamic inactivation (PDI) was shown to be a promising treatment modality for microbial infections. This study explores the effect of chitosan, a polycationic biopolymer, in increasing the PDI efficacy against Gram-positive bacteria, including Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, and methicillin-resistant S. aureus (MRSA), as well as the Gram-negative bacteria Pseudomonas aeruginosa and Acinetobacter baumannii. Chitosan at <0.1% was included in the antibacterial process either by coincubation with hematoporphyrin (Hp) and subjection to light exposure to induce the PDI effect or by addition after PDI and further incubation for 30 min. Under conditions in which Hp-PDI killed the microbe on a 2- to 4-log scale, treatment with chitosan at concentrations of as low as 0.025% for a further 30 min completely eradicated the bacteria (which were originally at ∼108 CFU/ml). Similar results were also found with toluidine blue O (TBO)-mediated PDI in planktonic and biofilm cells. However, without PDI treatment, chitosan alone did not exert significant antimicrobial activity with 30 min of incubation, suggesting that the potentiated effect of chitosan worked after the bacterial damage induced by PDI. Further studies indicated that the potentiated PDI effect of chitosan was related to the level of PDI damage and the deacetylation level of the chitosan. These results indicate that the combination of PDI and chitosan is quite promising for eradicating microbial infections.

Use of Merocyanine 540 for Photodynamic Inactivation of Staphylococcus aureus Planktonic and Biofilm Cells

ABSTRACT Photodynamic inactivation of Staphylococcus aureus planktonic and biofilm cells by a phtotosensitizer, merocyanine 540 (MC 540), was investigated. For the planktonic experiments, MC 540 binding efficiency to bacterial cells was found to increase with both increasing MC 540 concentration and increasing incubation time, but the binding became saturated following 10 min of incubation. The antimicrobial activity was enhanced with an increasing light dose, but an increase in the light dose could not further improve the antimicrobial activity if the maximum excitation level attainable was less than the necessary minimum threshold level. Complete inactivation was achieved when the excitation level of MC 540 was somewhere above the threshold level. The relationship between antimicrobial activity and the excitation level of MC 540 revealed that the more MC 540 was excited, the more S. aureus cells were killed. For the biofilm experiments, the antimicrobial activity was enhanced with an increase in the light dose. No viable cells were detected when organisms were exposed to 15 μg of MC 540 per ml and a light dose of 600 J/cm2 or to 20 μg of MC 540 per ml and a light dose of 450 J/cm2. A quantitative analysis of MC 540 bound to biofilms was also performed, and the images from confocal laser scanning microscopy provided direct evidence that revealed the difference between the MC 540 remaining in the biofilms prior to irradiation and the MC 540 remaining in the biofilms after irradiation. The results of both the planktonic and biofilm experiments suggest that the antimicrobial activity of photodynamic inactivation of S. aureus is closely related to the excitation level of MC 540.

Reishi immuno-modulation protein induces interleukin-2 expression via protein kinase-dependent signaling pathways within human T cells†

Ganoderma lucidum, a medicinal fungus is thought to possess and enhance a variety of human immune functions. An immuno‐modulatory protein, Ling Zhi‐8 (LZ‐8) isolated from G. lucidum exhibited potent mitogenic effects upon human peripheral blood lymphocytes (PBL). However, LZ‐8‐mediated signal transduction in the regulation of interleukin‐2 (IL‐2) gene expression within human T cells is largely unknown. Here we cloned the LZ‐8 gene of G. lucidum, and expressed the recombinant LZ‐8 protein (rLZ‐8) by means of a yeast Pichia pastoris protein expression system. We found that rLZ‐8 induces IL‐2 gene expression via the Src‐family protein tyrosine kinase (PTK), via reactive oxygen species (ROS), and differential protein kinase‐dependent pathways within human primary T cells and cultured Jurkat T cells. In essence, we have established the nature of the rLZ‐8‐mediated signal‐transduction pathways, such as PTK/protein kinase C (PKC)/ROS, PTK/PLC/PKCalpha/ERK1/2, and PTK/PLC/PKCalpha/p38 pathways in the regulation of IL‐2 gene expression within human T cells. Our current results of analyzing rLZ‐8‐mediated signal transduction in T cells might provide a potential application for rLZ‐8 as a pharmacological immune‐modulating agent. J. Cell. Physiol. 215: 15–26, 2008.

Compatibility balanced antibacterial modification based on vapor-deposited parylene coatings for biomaterials

Advanced antibacterial surfaces are designed based on covalently attached antibacterial agents, avoiding potential side effects associated with overdosed or eluted agents. The technique is widely applicable regardless of the underlying substrate material. In addition, antibacterial surfaces are effective against the early stages of bacterial adhesion and can significantly reduce the formation of biofilm, without compromising biocompatibility. Here, this concept was realized by employing a benzoyl-functionalized parylene coating. The antibacterial agent chlorhexidine was used as a proof of concept. Chlorhexidine was immobilized by reaction with photoactivated benzoyl-functionalized surfaces, including titanium alloy, stainless steel, polyether ether ketone, polymethyl methacrylate, and polystyrene. A low concentration of chlorhexidine (1.4 ± 0.08 nmol cm-2) covalently bound to surfaces rendered them sufficiently resistant to an Enterobacter cloacae inoculum and its adherent biofilm. Compared to unmodified surfaces, up to a 30-fold reduction in bacterial attachment was achieved with this coating technology. The immobilization of chlorhexidine was verified with infrared reflection absorption spectroscopy (IRRAS) and X-ray photoelectron spectroscopy (XPS), and a leaching test was performed to confirm that the chlorhexidine molecules were not dislodged. Cell compatibility was examined by culturing fibroblasts and osteoblasts on the modified surfaces, revealing greater than 93% cell viability. This coating technology may be broadly applicable for a wide range of other antibacterial agents and allow the design of new biomaterials.

Effects of ultrasonic treatment on the efficacy of gentamicin against established Pseudomonas aeruginosa biofilms

Abstract The effect of simultaneous ultrasonic treatment on the efficacy of gentamicin against planktonic and established biofilm cells of Pseudomonas aeruginosa was investigated. Planktonic cells were treated with 6 or 12 μg ml−1 of gentamicin for 4 h with ultrasonic treatment at three levels of power density (0.2, 2 and 15 mW cm−2). Biofilm cells grown on stainless steel slides in a continuous flow reactor were treated with 30 μg ml−1 of gentamicin and ultrasound. Ultrasound itself at these power levels did not cause cell killing or lysis in planktonic and biofilm cultures. Concentrations of 6 and 12 mg ml−1 gentamicin led to 2.65- and 2.75-log reductions of the surviving fraction in planktonic cultures in the absence of ultrasound. The addition of ultrasound did not show further reduction compared with those without ultrasonication. Gentamicin (30 μg ml−1) caused variable killing in biofilms which ranged from 0.83- to 2.86-log reductions of the surviving fraction without ultrasonication. Gentamicin efficacy measured by the surviving fraction was improved by 0.28-, 1.12- and 0.58-log when coupled with 0.2, 2 and 15 mW cm−2 ultrasonic treatments, respectively. Experimental results indicated that ultrasound modestly improved the efficacy of gentamicin against established P. aeruginosa biofilms.

Use of flow cell reactors to quantify biofilm formation kinetics

A parallel plate flow cell reactor is introduced and used to evaluate cell adhesion and biofilm formation kinetics for four different bacterial strains of the species,E. coli. The reactor allows biofilm growth under defined, well-controlled fluid dynamics while providing continuous observations and direct sampling of biofilm for biological, chemical and physical analyses as well as immunofluorescent labeling.