Browsing by Author "Huang, Ching-Tsan"

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Biofilm control by antimicrobial agents
(2000) Stewart, Philip S.; McFeters, Gordon A.; Huang, Ching-Tsan
Effects of inducer levels on a recombinant bacterial biofilm formation and gene expression
(1994-09) Huang, Ching-Tsan; Peretti, Steven W.; Bryers, James D.
A segregationally stable host-plasmid system, E. coli DH5α (pTKW106), was used to study the effect of induction on the accumulation rate of cells and gene expression in biofilm cultures. Isopropyl β-D-thiogalactoside (IPTG) was used to induce the expression of β-galactosidase from the plasmid. The biofilm cell net accumulation rates decreased with increasing induction levels. At 0.17 and 0.34 mM of IPTG, the biofilm cell net accumulation rates ranged between 17 and 30% when compared to the uninduced case. At 0.51 mM of IPTG, the biofilm cell density never increased. At 0.17 and 0.34 mM of IPTG, β-galactosidase contents reached maxima 36 hours after induction with both amounts representing about 7.5% of total protein. At 0.51 mM of IPTG, β-galactosidase production reached its maximum, about 16% of total protein, 48 hours after induction. The β-galactosidase mRNA synthesis rates increased with increasing inducer levels. Maximum β-galactosidase mRNA synthesis rates were reached 36 hours after induction for each IPTG concentration.
Effects of medium carbon-to-nitrogen ratio on biofilm formation and plasmid stability
(1994-07) Huang, Ching-Tsan; Peretti, Steven W.; Bryers, James D.
Biofilm formation and plasmid segregational instability in biofilm cultures of Escherichia coli DH5α (pMJR1750) were investigated under different medium-carbon-to-nitrogen (C/N) ratios. At C/N ratios of 0.07 and 1, net accumulation of both biofilm plasmid-bearing and plasmid-free cells continued through the entire experiment without attaining any apparent steady state. At C/N ratios of 5 and 10, net biofilm cell accumulation for the two populations reached apparent steady states after 84 and 72 h, respectively. At C/N ratios of 0.07 and 1, polysaccharide production increased slowly and reached about 2g alginate equivalent/cm2 by the end of both experiments. At a C/N ratio of 5, polysaccharide increase significantly after 84 h, reaching about 7μg alginate equivalent/cm2 prior to termination. At a C/N ratio of 10, polysaccharide increased significantly after 72 h and reached 21 μg alginate equivalent/cm2 at 108 h. At C/N ratios of 0.07 and 1, protein production reached 6.5 and 4 μg/cm2, respectively. At C/N ratios of 5 and 10, protein production increased slightly for the first 84 h and reached a maximum at 108 h, at 3 and 2 μg/cm2, respectively, then decreased over the last 12 h of the experiment. Ratios of polysaccharide to protein increased with increasing C/N ratios. At C/N ratios of 0.07 and 1, the ratios between extracellular polysaccharide (EP) and protein were no more than 205 μg polysaccharide/μg protein, whereas those at C/N ratios of 5 and 10 increased to about 7 and 12 μg polysaccharide/μg protein, respectively. Probabilities of plasmid loss in the biofilm cultures increased with increasing C/N ratios. At C/N ratios of 0.07, 1, and 5, the probabilities of plasmid loss were 0.0013 ± 0.011, 0.020 ± 0.006 and 0.122 ± 0.021, respectively. At a C/N ratio of 10, the probability of plasmid loss was significantly higher, reaching 0.38 ± 0.125. The increase of probability of plasmid loss at higher C/N ratios results from competition between cell replication and extracellular polysaccharide production. © 1994 John Wiley & Sons, Inc.
Effects of ultrasonic treatment on the efficacy of gentamicin against established pseudomonas aeruginosa biofilms
(1996-05) Huang, Ching-Tsan; James, Garth A.; Pitt, William G.; Stewart, Philip S.
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.
Evaluation of physiological staining, cryoembedding and autofluorescence quenching techniques on fouling biofilms
(1996-07) Huang, Ching-Tsan; McFeters, Gordon A.; Stewart, Philip S.
Physiological staining, cryoembedding, cryosectioning and autoftuorescence quenching techniques were evaluated for their applicability to undefined mixed population biofilms collected from environmental or engineered systems. Four different biofilms from two cooling towers, a paper mill machine and the effluent ditch of a wastewater treatment plant were tested. The redox dye 5‐cyano‐2,3‐ditolyl tetrazolium chloride (CTC) was used in combination with the DNA stain 4’,6‐diamino‐2‐phenylindole (DAPI) to distinguish respiring and nonrespiring cells. Positive CTC staining, as evidenced by the development of pink or red color, was successful in all samples examined except for paper mill biofilm. The structural integrity of frozen sections deteriorated when biofilms contained rigid or fibrous material. Autofluorescence generally impaired the ability to distinguish specific staining from natural background fluorescence. Two physical and three chemical methods were tested to quench autofluorescence. Quenching with crystal violet reduced most of the autofluorescent interference and still maintained physiological staining intensity, but contrast between CTC staining and residual autofluorescence was poor. Autofluorescence and the difficulty of sectioning thick biofilms containing abiotic materials limit the applicability of cryoembedding/staining techniques to fouling biofilms.
Nonuniform spatial patterns of respiratory activity within biofilms during disinfection
(1995-06) Huang, Ching-Tsan; Yu, Feipeng Philip; McFeters, Gordon A.; Stewart, Philip S.
Fluorescent stains in conjunction with cryoembedding and image analysis were applied to demonstrate spatial gradients in respiratory activity within bacterial biofilms during disinfection with monochloramine. Biofilms of Klebsiella pneumoniae and Pseudomonas aeruginosa grown together on stainless steel surfaces in continuous-flow annular reactors were treated with 2 mg of monochloramine per liter (influent concentration) for 2 h. Relatively little biofilm removal occurred as evidenced by total cell direct counts. Plate counts (of both species summed) indicated an average 1.3-log decrease after exposure to 2 mg of monochloramine per liter. The fluorogenic redox indicator 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and the DNA stain 4*,6-diamidino2-phenylindole (DAPI) were used to differentiate respiring and nonrespiring cells in biofilms. Epifluorescence micrographs of frozen biofilm cross sections clearly revealed gradients of respiratory activity within biofilms in response to monochloramine treatment. These gradients in specific respiratory activity were quantified by calculating the ratio of CTC and DAPI intensities measured by image analysis. Cells near the biofilm-bulk fluid interface lost respiratory activity first. After 2 h of biocide treatment, greater respiratory activity persisted deep in the biofilm than near the biofilm-bulk fluid interface.
Physiological heterogeneity and biofilm control," in: biofilms in the aquatic environment
(1999) McFeters, Gordon A.; Stewart, Philip S.; Huang, Ching-Tsan; Wentland, Eric Jon; Xu, Karen D.; Yu, Feipeng Philip
Plasmid retention and cloned gene expression in suspended versus biofilm cultures of e. coli dh5(pmjr1750)
(1993-01) Huang, Ching-Tsan; Peretti, Steven W.; Bryers, James D.
Differences in plasmid retention and expression are studied in both suspended and biofilm cultures of Escherichia coli DH5α(PMJR1750). An alternative mathematical model is proposed which allows the determination of plasmid loss probability in both suspended batch and continuously fed biofilm cultures. In our experiments, the average probability of plasmid loss of E. coli DH5α(pMJR1750) is 0.0022 in batch culture in the absence of antibiotic selection pressure and inducer. Under the induction of 0.17 MM IPTG, the maximum growth rate of plasmid-bearing cells in suspended batch culture dropped from 0.45 h−1 to 0.35 h−1 and the β-galactosidase concentration reached an experimental maximum of 0.32. pg/cell 4 hours after the initiation of induction. At both 0.34 and 0.51 mM IPTG, growth rates in batch cultures decreased to 0.16 h−1, about 36% of that without IPTG, and the β-galactosidase concentration reached an experimental maximum of 0.47 pg/cell 3 hours after induction. In biofilm cultures, both plasmid-bearing and plasmid-free cells in increase with time reaching a plateau after 96 hours n the absence of both the inducer and any antibiotic selection pressure. Average probability of plasmid loss for biofilm-bound E. coli DH5β(pMJR1750) population was 0.017 without antibiotic selection. Once the inducer IPTG was added, the concentration of plasmid-bearing cells in biofilm dropped dramatically while plasmid-free cell numbers maintained unaffected. The β-galactosidase concentration reached a maximum in all biofilm experiments 24 hours after induction; they were 0.08, 0.1, and 0.12 pg/cel under 0.17, 0.34, and 0.51 mM IPTG, respectively.
Quorum sensing in pseudomonas aeruginosa controls expression of catalase and superoxide dismutase genes and mediates biofilm susceptibility to hydrogen peroxide
(1999-12) Hassett, Daniel J.; Ma, J.-F.; Elkins, James D.; McDermott, Timothy R.; Ochsner, Urs A.; West, Susan E. H.; Huang, Ching-Tsan; Fredericks, J.; Burnett, S.; Stewart, Philip S.; McFeters, Gordon A.; Passador, L.; Iglewski, Barbara H.
Quorum sensing (QS) governs the production of virulence factors and the architecture and sodium dodecyl sulphate (SDS) resistance of biofilm-grown Pseudomonas aeruginosa. P. aeruginosaQS 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.
Recombinant plasmid retention and expression in bacterial biofilm cultures
(1995) Bryers, James D.; Huang, Ching-Tsan
Any exposure of plasmid recombinant microorganisms to an open system environment, either inadvertently or intentionally, mandates research into those fundamental organism:plasmid processes that influence plasmid retention, transfer and expression. In open environmental systems a majority of the microbial activity occurs associated with an interface, within thin biological layers consisting of the cells and their insoluble extracellular polymer, layers known as biofilms. Thus any study regarding the fate of recombinant DNA sequences in an open system must consider processes that affect plasmid retention and expression in a biofilm culture. Biofilm cultures were cultivated in a parallel-plate flow cell reactor using E. coli DH5α which contained a recombinant plasmid with a plasmid stability factor, parB, (pTKW106) or without (pMJR1750). Using β-galactosidase as inducible reporter protein, plasmid retention and gene expression of pMJR1750 and pTKW106, in suspended versus biofilm cultures, were studied under different carbo to nitrogen ratios and plasmid induction levels. Recombinant biofilm formation under these environmental conditions was also investigated. Biofilm net accumulation rate of E. coli DH5α (pTKW106) decreases with increasing induction levels. The β-galactosidase production and ratios of β-galactosidase to total protein increase with increasing induction levels. Synthesis rates of total RNA, β-galactosidase mRNA and rRNA in biofilm cultures of E. coli DH5α (pTKW106) increase after induction by IPTG.
Reduction of polysaccharide production in pseudomonas aeruginosa biofilms by bismuth dimercaprol (bisbal) treatment
(1999-11) Huang, Ching-Tsan; Stewart, Philip S.
Microorganisms in biofilms, cells attached to a surface and embedded in secreted insoluble extracellular polymers, are recalcitrant to chemical biocides and antibiotics. When Pseudomonas aeruginosa ERC1 biofilms were treated continuously with 1 × MIC of bismuth dimercaprol (BisBAL), biofilm density determined by both total cell counts and viable cell counts increased during the first 30 h period then decreased thereafter. After 120 h of treatment there was an approximate 3-log reduction in viable cell areal density compared with the untreated control. Per-cell total polysaccharide production was significantly reduced in biofilms exposed to 12.5 μM BisBAL compared with the untreated control. In biofilm cultures, 1 × MIC of BisBAL did not initially kill attached cells but was enough to reduce polysaccharide production. As treatment proceeded, the normalized polysaccharide content was reduced and those cells attached became susceptible to 1 × MIC of BisBAL.
Spatial distribution and coexistence of klebsiella pneumoniae and pseudomonas aeruginosa in biofilms
(1997-01) Stewart, Philip S.; Camper, Anne K.; Handran, S. D.; Huang, Ching-Tsan; Warnecke, Malcolm R.
The 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.
Spatial patterns of alkaline phosphatase expression within bacterial colonies and biofilms in response to phosphate starvation
(1998-04) Huang, Ching-Tsan; Xu, Karen D.; McFeters, Gordon A.; Stewart, Philip S.
The expression of alkaline phosphatase in response to phosphate starvation was shown to be spatially and temporally heterogeneous in bacterial biofilms and colonies. A commercial alkaline phosphatase substrate that generates a fluorescent, insoluble product was used in conjunction with frozen sectioning techniques to visualize spatial patterns of enzyme expression in both Klebsiella pneumoniae and Pseudomonas aeruginosa biofilms. Some of the expression patterns observed revealed alkaline phosphatase activity at the boundary of the biofilm opposite the place where the staining substrate was delivered, indicating that the enzyme substrate penetrated the biofilm fully. Alkaline phosphatase accumulated linearly with time in K. pneumoniae colonies transferred from high-phosphate medium to low-phosphate medium up to specific activities of 50 μmol per min per mg of protein after 24 h. In K. pneumoniae biofilms and colonies, alkaline phosphatase was initially expressed in the region of the biofilm immediately adjacent to the carbon and energy source (glucose). In time, the region of alkaline phosphatase expression expanded inward until it spanned most, but not all, of the biofilm or colony depth. In contrast, expression of alkaline phosphatase in P. aeruginosa biofilms occurred in a thin, sharply delineated band at the biofilm-bulk fluid interface. In this case, the band of activity never occupied more than approximately one-sixth of the biofilm. These results are consistent with the working hypothesis that alkaline phosphatase expression patterns are primarily controlled by the local availability of either the carbon and energy source or the electron acceptor.
Spatial physiological heterogeneity in Pseudomonas aeruginosa biofilm is determined by oxygen availability
(1998-10) Xu, Karen D.; Stewart, Philip S.; Xia, Fuhu; Huang, Ching-Tsan; McFeters, Gordon A.
Spatial variations in growth rate within Klebsiella pneumoniae colonies and biofilm
(1996-06) Wentland, Eric Jon; Stewart, Philip S.; Huang, Ching-Tsan; McFeters, Gordon A.
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.
The study of microbial biofilms by classical fluorescence microscopy
(1998) Huang, Ching-Tsan; Stewart, Philip S.; McFeters, Gordon A.
Use of flow cell reactors to quantify biofilm formation kinetics
(1992-05) Huang, Ching-Tsan; Peretti, Steven W.; Bryers, James D.
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.