Browsing by Author "Pitts, Betsey"

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Antimicrobial activity of synthetic cationic peptides & lipopeptides derived from human lactoferricin against Pseudomonas aeruginosa planktonic cultures and biofilms
(2015-07) Sánchez-Gómez, Susana; Ferrer-Espada, Raquel; Stewart, Philip S.; Pitts, Betsey; Lohner, Karl; Martinez de Tejada, Guillermo
Background Infections by Pseudomonas aeruginosa constitute a serious health threat because this pathogen - particularly when it forms biofilms - can acquire resistance to the majority of conventional antibiotics. This study evaluated the antimicrobial activity of synthetic peptides based on LF11, an 11-mer peptide derived from human lactoferricin against P. aeruginosa planktonic and biofilm-forming cells. We included in this analysis selected N-acylated derivatives of the peptides to analyze the effect of acylation in antimicrobial activity. To assess the efficacy of compounds against planktonic bacteria, microdilution assays to determine the minimal inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time-kill studies were conducted. The anti-biofilm activity of the agents was assessed on biofilms grown under static (on microplates) and dynamic (in a CDC-reactor) flow regimes. Results The antimicrobial activity of lipopeptides differed from that of non-acylated peptides in their killing mechanisms on planktonic and biofilm-forming cells. Thus, acylation enhanced the bactericidal activity of the parental peptides and resulted in lipopeptides that were uniformly bactericidal at their MIC. In contrast, acylation of the most potent anti-biofilm peptides resulted in compounds with lower anti-biofilm activity. Both peptides and lipopeptides displayed very rapid killing kinetics and all of them required less than 21 min to reduce 1,000 times the viability of planktonic cells when tested at 2 times their MBC. The peptides, LF11-215 (FWRIRIRR) and LF11-227 (FWRRFWRR), displayed the most potent anti-biofilm activity causing a 10,000 fold reduction in cell viability after 1 h of treatment at 10 times their MIC. At that concentration, these two compounds exhibited low citotoxicity on human cells. In addition to its bactericidal activity, LF11-227 removed more that 50 % of the biofilm mass in independent assays. Peptide LF11-215 and two of the shortest and least hydrophobic lipopeptides, DI-MB-LF11-322 (2,2-dimethylbutanoyl-PFWRIRIRR) and DI-MB-LF11-215, penetrated deep into the biofilm structure and homogenously killed biofilm-forming bacteria. Conclusion We identified peptides derived from human lactoferricin with potent antimicrobial activity against P. aeruginosa growing either in planktonic or in biofilm mode. Although further structure-activity relationship analyses are necessary to optimize the anti-biofilm activity of these compounds, the results indicate that lactoferricin derived peptides are promising anti-biofilm agents."
Antimicrobial penetration and efficacy in an in vitro oral biofilm model
(2011-05) Corbin, A.; Pitts, Betsey; Parker, Albert E.; Stewart, Philip S.
The penetration and overall efficacy of six mouthrinse actives was evaluated by using an in vitro flow cell oral biofilm model. The technique involved preloading biofilm cells with a green fluorescent dye that leaked out as the cells were permeabilized by a treatment. The loss of green color, and of biomass, was observed by time-lapse microscopy during 60 min of treatment under continuous flow conditions. The six actives analyzed were ethanol, sodium lauryl sulfate, triclosan, chlorhexidine digluconate (CHX), cetylpyridinium chloride, and nisin. Each of these agents effected loss of green fluorescence throughout biofilm cell clusters, with faster action at the edge of a cell cluster and slower action in the cluster center. The time to reach half of the initial fluorescent intensity at the center of a cell cluster, which can be viewed as a combined penetration and biological action time, ranged from 0.6 to 19 min for the various agents. These times are much longer than the predicted penetration time based on diffusion alone, suggesting that anti-biofilm action was controlled more by the biological action time than by the penetration time of the active. None of the agents tested caused any removal of the biofilm. The extent of fluorescence loss after 1 h of exposure to an active ranged from 87 to 99.5%, with CHX being the most effective. The extent of fluorescence loss in vitro, but not penetration and action time, correlated well with the relative efficacy data from published clinical trials.
Antimicrobial-coated endotracheal tubes: An experimental study
(2008-04) Berra, Lorenzo; Curto, Francesco; Bassi, Gianluigi L.; Laquerriere, Patrice; Pitts, Betsey; Baccarelli, Andrea; Kolobow, Theodor
OBJECTIVE: Antibiotic-resistant bacterial biofilm may quickly form on endotracheal tubes (ETTs) and can enter the lungs, potentially causing pneumonia. In an attempt to prevent bacterial colonization, we developed and tested in an in-vitro study and animal study several antibacterial-coated ETTs (silver sulfadiazine with and without carbon in polyurethane, silver sulfadiazine and chlorhexidine with and without carbon in polyurethane, silver-platinum with and without carbon in polyurethane, chlorhexidine in polyurethane, and rose bengal for UV light). DESIGN, SETTING, ANIMALS, INTERVENTIONS: After preliminary studies, silver sulfadiazine in polyurethane (SSD-ETT) was selected among the coatings to be challenged every 24h with 104-106 Pseudomonas aeruginosa/ml and evaluated at 6h, 24h, and 72h with standard microbiological studies, scanning electron microscopy, and confocal scanning microscopy. Subsequently, eight sheep were randomized to receive either a SSD-ETT or a standard ETT (St-ETT). After 24h of mechanical ventilation, standard microbiological studies were performed together with scanning electron microscopy and confocal microscopy. MEASUREMENTS AND RESULTS: In the in-vitro study SSD-ETT remained bacteria-free for up to 72h, whereas St-ETT showed heavy P. aeruginosa growth and biofilm formation (p < 0.01). In sheep, the SSD-ETT group showed no bacterial growth in the ETT, ventilator tubing, and lower respiratory tract, while heavy colonization was found in the St-ETT (p < 0.01), ventilator tubing (p = 0.03), and lower respiratory tract (p < 0.01). CONCLUSION: This study describes several effective and durable antibacterial coatings for ETTs. Particularly, SSD-ETT showed prevention against P. aeruginosa biofilm formation in a 72-h in-vitro study and lower respiratory tract colonization in sheep mechanically ventilated for 24h.
Bacterial characterization of toilet bowl biofilms
(1998-08) Pitts, Betsey; Stewart, Philip S.; McFeters, Gordon A.; Hamilton, Martin A.; Willse, Alan Ray; Zelver, Nick
Methods have been developed and applied for sampling, characterizing and quantifying naturally occurring toilet bowl biofilms. Ceramic porcelain disks mounted in neoprene rubber strips were sealed in place in toilet bowls in three residences in Bozeman, Montana. In each bowl, duplicate strips were placed above, at and below the water level. In 7 consecutive weeks, duplicate disks from each zone in each bowl were removed. Surface biofouling was measured by viable cell areal density. Specific fouling rates were calculated and variability among toilet bowls and water levels was assessed. Specific fouling rates ranged from 0.0 to 0.46d‐1. Average areal cell densities at the end of 7 weeks ranged from 103 to 107cfu cm‐2. The extent of fouling was highest below the water line. Neutralization of the chlorine residual (typically 0.9 mg l‐1) in one toilet did not increase the extent of fouling compared to the controls. Biofilm areal viable cell densities and bowl water viable counts were positively correlated (r = 0.78). The visual threshold for detection of toilet bowl biofilm by the naked eye was approximately 105 cfu cm‐2. In a heavily fouled toilet bowl, the biofilm was up to 20 μm thick. Microorganisms were isolated from the biofilm and identified. Of the 32 organisms that were further characterized, 10 were identified as Pseudomonas, Sphingomonas or Chryseomonas species.
Characterization of a modified rotating disk reactor for the cultivation of Staphylococcus epidermidis biofilm
(2010-09) Cotter, John J.; O'Gara, James P.; Pitts, Betsey; Casey, Eoin
Aims: The purpose of this study was to develop a system that would allow biofilms to be cultivated under strictly defined conditions in terms of dissolved oxygen, fluid shear and to assess whether the method was suitable for the detection of respiratory activity stratification in biofilm samples.Methods:â€‚The system is a modified version a commercially available laboratory biofilm reactor and incorporates a number of features such as the provision of defined levels of dissolved oxygen, constant average shear, enhanced gasâ€“liquid mass transfer, aseptic operation and the ability to remove biofilm for ex situ analysis during or after continuous cultivation.Conclusions:â€‚The system was shown to be effective for the characterization of the effects of dissolved oxygen on a pure culture of Staphylococcus epidermidis. The versatility of the system offers the potential for cultivating pure culture biofilm in defined, controlled conditions and facilitates a range of analyses that can be performed ex situ.Significance and Impact of the Study: The ability to provide strict regulation of environmental conditions and enhanced transfer of oxygen to the biofilm during cultivation are important, first because oxygen is known to regulate biofilm development in several microorganisms and second because many conventional biofilm cultivation systems may not provide adequate oxygen supply to the biofilm.
Color measurement as a means of quantifying surface biofouling
(1998-11) Pitts, Betsey; Hamilton, Martin A.; McFeters, Gordon A.; Stewart, Philip S.; Willse, Alan Ray; Zelver, Nick
Laboratory reactors fitted with removable ceramic porcelain growth surfaces were inoculated with a consortium of biofilm forming environmental isolates. A Minolta colorimeter CR-200 (Minolta Camera Co., Ltd, Ramsey, NJ) was used in conjunction with a specially designed adapter to evaluate the reflective color of the porcelain disks as biofilm accumulated on them. Areal viable cell counts were monitored over a period of eleven days in two separate experiments and direct color measurements of the untreated, microbially fouled test surfaces were collected. This colorimetric assay was both non-destructive and immediate. A strong linear relationship between log cell density and log color change was observed. The Pearson product moment correlation coefficient for all 45 observations combined was r=0.95. Separate regression lines for each experiment were not significantly different (P=0.19). When adjusted for time, the (partial) correlation coefficient between log cell density and log color change was r=0.87, which suggests that the relationship between the two measures can not be explained by their mutual dependence on time. Reflective color measurement provided a rapid, non-destructive and quantitative measure of biofilm accumulation.
Comparison of antimicrobial effect on biofilm of chlorine, silver ion and tobramycin
(2008-01) Kim, Jaeeun; Pitts, Betsey; Stewart, Philip S.; Camper, Anne K.; Yoon, Jeyong
The systematic understanding of how various antimicrobial agents are involved in controlling biofilm is essential in order to establish an effective strategy for biofilm control, since many antimicrobial agents are effective against planktonic cells but ineffective when used against the same bacteria growing in a biofilm state. Three different antimicrobial agents (chlorine, silver, and tobramycin) and three different measures of efficacy were selected to examine antimicrobial activity on biofilm in a comparative way: plate counts, measurement of respiratory activity with CTC staining, and BacLight Live/Dead stain for measuring membrane integrity were used along with CLSM and epifluorescence microscopy. The three methods of determining antimicrobial activity gave very different results for each antimicrobial agent. Tobramycin appears to be the most effective in reducing the respiratory activity of biofilm cells compared to chlorine and silver, based upon CTC staining. In contrast, tobramycin-treated biofilm cells maintained membrane integrity better than chlorine or silver treated ones, evidenced by both CLSM and epifluorescence microscope imaging. Combined and sequential treatments with silver and tobramycin showed an enhanced antimicrobial efficiency of more than 200%, while the antimicrobial activity of either chlorine or tobramycin was antagonized when the agents were used in combination. This observation makes sense when the differing oxidative reactivities of chlorine, silver and tobramycin are considered.
Confocal laser microscopy on biofilms: Successes and limitations
(2008-07) Pitts, Betsey; Stewart, Philip S.
Imaging of bacterial biofilms with microscopes has been an essential and transformative method in biofilm research. Fluorescence microscopy can elucidate specific biofilm components and cellular activities that cannot be separated otherwise. In particular, confocal fluorescence microscopy extends that examination through the thickness of a fully hydrated, in-situ biofilm, affording the potential for 3D, non-invasive, time-lapse imaging. This article discusses some striking examples of the insight provided by confocal fluorescence microscopy into biofilm structure, composition, and heterogeneity, and will also enumerate some limitations of this imaging process.
Development of a laboratory model of a phototroph-heterotroph mixed-species biofilm at the stone/air interface
(2015-11) Villa, Federica; Pitts, Betsey; Lauchnor, Ellen G.; Cappitelli, Francesca; Stewart, Philip S.
Recent scientific investigations have shed light on the ecological importance and physiological complexity of subaerial biofilms (SABs) inhabiting lithic surfaces. In the field of sustainable cultural heritage (CH) preservation, mechanistic approaches aimed at investigation of the spatiotemporal patterns of interactions between the biofilm, the stone, and the atmosphere are of outstanding importance. However, these interactions have proven difficult to explore with field experiments due to the inaccessibility of samples, the complexity of the ecosystem under investigation and the temporal resolution of the experiments. To overcome these limitations, we aimed at developing a unifying methodology to reproduce a fast-growing, phototroph-heterotroph mixed species biofilm at the stone/air interface. Our experiments underscore the ability of the dual-species SAB model to capture functional traits characteristic of biofilms inhabiting lithic substrate such as: (i) microcolonies of aggregated bacteria; (ii) network like structure following surface topography; (iii) cooperation between phototrophs and heterotrophs and cross feeding processes; (iv) ability to change the chemical parameters that characterize the microhabitats; (v) survival under desiccation and (vi) biocide tolerance. With its advantages in control, replication, range of different experimental scenarios and matches with the real ecosystem, the developed model system is a powerful tool to advance our mechanistic understanding of the stone-biofilm-atmosphere interplay in different environments.
Diffusion of Macromolecules in Model Oral Biofilms
(2009-01) Takenaka, Shoji; Pitts, Betsey; Trivedi, Harsh M.; Stewart, Philip S.
The diffusive penetration of fluorescently tagged macromolecular solutes into model oral biofilms was visualized by time-lapse microscopy. All of the solutes tested, including dextrans, proteases, green fluorescent protein, and immunoglobulin G, accessed the interior of cell clusters 100 to 200 μm in diameter within 3 min or less.
Diffusive transport through a model host-biofilm system
(2015-08) Aristotelous, A. C.; Klapper, Isaac; Grabovsky, Y.; Pabst, Breana; Pitts, Betsey; Stewart, Philip S.
Free-living biofilms have been subject to considerable attention, and basic physical principles for them are generally accepted. Many host-biofilm systems, however, consist of heterogeneous mixtures of aggregates of microbes intermixed with host material and are much less studied. Here we analyze a key property, namely reactive depletion, in such systems and argue that two regimes are possible: (1) a homogenizable mixture of biofilm and host that in important ways acts effectively like a homogeneous macrobiofilm and (2) a distribution of separated microbiofilms within the host with independent local microenvironments.
Direct visualization of spatial and temporal patterns of antimicrobial action within model oral biofilms
(2008-01) Takenaka, Shoji; Trivedi, Harsh M.; Corbin, A.; Pitts, Betsey; Stewart, Philip S.
A microscopic method for non-invasively visualizing the action of an antimicrobial agent inside a biofilm was developed and applied to describe spatial and temporal patterns of mouthrinse activity on model oral biofilms. Three species biofilms of Streptococcus oralis, Streptococcus gordonii, and Actinomyces naeslundii were grown in glass capillary flow cells. Bacterial cells were stained with the fluorogenic esterase substrate Calcien AM. Loss of green fluorescence upon exposure to an antimicrobial formulation was subsequently imaged by time-lapse confocal laser scanning microscopy. When an antimicrobial mouthrinse containing chlorhexidine digluconate was administered, a gradual loss of green fluorescence was observed that began at the periphery of cell clusters where they adjoined the flowing bulk fluid and progressed inward over a time period of several minutes. Image analysis was performed to quantify a penetration velocity of 4 microns per minute. An enzyme-based antimicrobial formulation lead to a gradual, continually slowing loss of fluorescence in a pattern that was qualitatively different from the behavior observed with chlorhexidine. Ethanol at 11.6% had little effect on the biofilm. None of these treatments resulted in removal of biomass from the biofilm. Most methods to measure or visualize antimicrobial action in biofilms are destructive. Spatial information is important because biofilms are known for their structural and physiological heterogeneity. The CAM staining technique has the potential to provide information about the rate of antimicrobial penetration, the presence of tolerant subpopulations, and the extent of biomass removal effected by a treatment.
Gel-Entrapped Staphylococcus aureus Bacteria as Models of Biofilm Infection Exhibit Growth in Dense Aggregates Oxygen Limitation, Antibiotic Tolerance, and Heterogeneous Gene Expression
(2016-08) Pabst, Breana; Pitts, Betsey; Lauchnor, Ellen G.; Stewart, Philip S.
An experimental model that mimicked the structure and characteristics of in vivo biofilm infections, such as those occurring in the lung or in dermal wounds where no biomaterial surface is present, was developed. In these infections, microbial biofilm forms as cell aggregates interspersed in a layer of mucus or host matrix material. This structure was modeled by filling glass capillary tubes with an agarose gel that had been seeded with Staphylococcus aureus bacteria and then incubating the gel biofilm in medium for up to 30 h. Confocal microscopy showed that the bacteria formed in discrete pockets distributed throughout the gel matrix. These aggregates enlarged over time and also developed a size gradient, with the clusters being larger near the nutrient- and oxygen-supplied interface and smaller at greater depths. Bacteria entrapped in gels for 24 h grew slowly (specific growth rate, 0.06 h−1) and were much less susceptible to oxacillin, minocycline, or ciprofloxacin than planktonic cells. Microelectrode measurements showed that the oxygen concentration decreased with depth into the gel biofilm, falling to values less than 3% of air saturation at depths of 500 μm. An anaerobiosis-responsive green fluorescent protein reporter gene for lactate dehydrogenase was induced in the region of the gel where the measured oxygen concentrations were low, confirming biologically relevant hypoxia. These results show that the gel biofilm model captures key features of biofilm infection in mucus or compromised tissue: formation of dense, distinct aggregates, reduced specific growth rates, local hypoxia, and antibiotic tolerance.
A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance
(2003-11) Mah, Thien-Fah; Pitts, Betsey; Pellock, Brett; Walker, Graham C.; Stewart, Philip S.; O'Toole, George A.
Biofilms are surface-attached microbial communities with characteristic architecture and phenotypic and biochemical properties distinct from their free-swimming, planktonic counterparts. One of the best-known of these biofilm-specific properties is the development of antibiotic resistance that can be up to 1,000-fold greater than planktonic cells. We report a genetic determinant of this high-level resistance in the Gram-negative opportunistic pathogen, Pseudomonas aeruginosa. We have identified a mutant of P. aeruginosa that, while still capable of forming biofilms with the characteristic P. aeruginosa architecture, does not develop high-level biofilm-specific resistance to three different classes of antibiotics. The locus identified in our screen, ndvB, is required for the synthesis of periplasmic glucans. Our discovery that these periplasmic glucans interact physically with tobramycin suggests that these glucose polymers may prevent antibiotics from reaching their sites of action by sequestering these antimicrobial agents in the periplasm. Our results indicate that biofilms themselves are not simply a diffusion barrier to these antibiotics, but rather that bacteria within these microbial communities employ distinct mechanisms to resist the action of antimicrobial agents.
Heterogeneity in Pseudomonas aeruginosa biofilms includes expression of ribosome hibernation factors in the antibiotic-tolerant subpopulation and hypoxia-induced stress response in the metabolically active population
(2012-02) Williamson, Kerry S.; Richards, Lee A.; Perez-Osorio, Ailyn C.; Pitts, Betsey; McInnerney, Kathleen; Stewart, Philip S.; Franklin, Michael J.
Bacteria growing in biofilms are physiologically heterogeneous, due in part to their adaptation to local environmental conditions. Here, we characterized the local transcriptome responses of Pseudomonas aeruginosa growing in biofilms by using a microarray analysis of isolated biofilm subpopulations. The results demonstrated that cells at the top of the biofilms had high mRNA abundances for genes involved in general metabolic functions, while mRNA levels for these housekeeping genes were low in cells at the bottom of the biofilms. Selective green fluorescent protein (GFP) labeling showed that cells at the top of the biofilm were actively dividing. However, the dividing cells had high mRNA levels for genes regulated by the hypoxia-induced regulator Anr. Slow-growing cells deep in the biofilms had little expression of Anr-regulated genes and may have experienced long-term anoxia. Transcripts for ribosomal proteins were associated primarily with the metabolically active cell fraction, while ribosomal RNAs were abundant throughout the biofilms, indicating that ribosomes are stably maintained even in slowly growing cells. Consistent with these results was the identification of mRNAs for ribosome hibernation factors (the rmf and PA4463 genes) at the bottom of the biofilms. The dormant biofilm cells of a P. aeruginosa Î”rmf strain had decreased membrane integrity, as shown by propidium iodide staining. Using selective GFP labeling and cell sorting, we show that the dividing cells are more susceptible to killing by tobramycin and ciprofloxacin. The results demonstrate that in thick P. aeruginosa biofilms, cells are physiologically distinct spatially, with cells deep in the biofilm in a viable but antibiotic-tolerant slow-growth state.
High-density targeting of a viral multifunctional nanoplatform to a pathogenic, biofilm-forming bacterium
(2007-04) Suci, Peter A.; Berglund, Deborah L.; Liepold, Lars Otto; Brumfield, Susan; Pitts, Betsey; Davison, William M.; Oltrogge, Luke; Hoyt, Kathryn Olivia; Codd, Sarah L.; Stewart, Philip S.; Young, Mark J.; Douglas, Trevor
Nanomedicine directed at diagnosis and treatment of infections can benefit from innovations that have substantially increased the variety of available multifunctional nanoplatforms. Here, we targeted a spherical, icosahedral viral nanoplatform to a pathogenic, biofilm-forming bacterium, Staphylococcus aureus. Density of binding mediated through specific protein-ligand interactions exceeded the density expected for a planar, hexagonally close-packed array. A multifunctionalized viral protein cage was used to load imaging agents (fluorophore and MRI contrast agent) onto cells. The fluorescence-imaging capability allowed for direct observation of penetration of the nanoplatform into an S. aureus biofilm. These results demonstrate that multifunctional nanoplatforms based on protein cage architectures have significant potential as tools for both diagnosis and targeted treatment of recalcitrant bacterial infections.
Identification of peptides derived from the human antimicrobial peptide LL-37 active against biofilms formed by Pseudomonas aeruginosa using a library of truncated fragments
(2012-11) Nagant, C.; Pitts, Betsey; Nazmi, K.; Vandenbranden, M.; Bolscher, J. G.; Stewart, Philip S.; Dehaye, J. P.
Persistent Pseudomonas aeruginosa infections are a major cause of morbidity and mortality in cystic fibrosis (CF) patients andare linked to the formation of a biofilm. The development of new biofilm inhibition strategies is thus a major challenge. LL-37 isthe only human antimicrobial peptide derived from cathelicidin. The effects on the P. aeruginosa PAO1 strain of synthetic truncatedfragments of this peptide were compared with the effects of the original peptide. Fragments of LL-37 composed of 19 residues(LL-19, LL13-31, and LL7-25) inhibited biofilm formation. The strongest antibiofilm activity was observed with the peptidesLL7-37 and LL-31, which decreased the percentage of biomass formation at a very low concentration. Some peptides werealso active on the bacteria within an established biofilm. LL7-31, LL-31, and LL7-37 increased the uptake of propidium iodide(PI) by sessile bacteria. The peptide LL7-37 decreased the height of the biofilm and partly disrupted it. The peptides active within the biofilm had an infrared spectrum compatible with an -helix. LL-37, but not the peptides LL7-31 and LL7-37, showed cellular toxicity by permeabilizing the eukaryotic plasma membrane (uptake of ethidium bromide and release of lactate dehydrogenase [LDH]). None of the tested peptides affected mitochondrial activity in eukaryotic cells. In conclusion, a 25-amino-acid peptide (LL7-31) displayed both strong antimicrobial and antibiofilm activities. The peptide was even active on cells within a preformed biofilm and had reduced toxicity toward eukaryotic cells. Our results also suggest the contribution of secondary structures ( -helix) to the activity of the peptides on biofilms.
Imaging biologically induced mineralization in fully hydrated flow systems
(2011) Schultz, Logan N.; Pitts, Betsey; Mitchell, Andrew C.; Cunningham, Alfred B.; Gerlach, Robin
A number of proposed technologies involve the controlled implementation of biologically induced carbonate mineral precipitation in the geologic subsurface. Examples include the enhancement of soil stability [1], immobilization of groundwater contaminants such as strontium and uranium [2], and the enhancement of oil recovery and geologic carbon sequestration via controlled permeability reduction [3]. The most significant challenge in these technologies remains to identify and better understand an industrially, environmentally, and economically viable carbonate precipitation route.One of the most promising routes is ureolytic biomineralization, because of the ample availability of urea and the controllable reaction rate. In this process, ureolytic bacteria hydrolyze urea, leading to an increase in pH. In the presence of calcium, this process favors the formation of solid calcium carbonate, as illustrated in the following equations:CO(NH2)2 + H2O â†’ NH2COOH + NH3â†’ 2 NH3 + CO2 (Urea hydrolysis) (1)2 NH3 + 2 H2O â†” 2NH4+ + 2OHâ€“ (pH increase) (2)CO2 + 2 OHâ€“ â†” CO32â€“ + H2O(Carbonate ion formation) (3)CO32â€“ + Ca2+ â†” CaCO3 (solid)(Precipitation is favored at high pH) (4)This process relies on molecular-level chemical and biological processes that must be better understood for large-scale implementation.Researchers at the Center for Biofilm Engineering at Montana State University (USA) and Aberystwyth University (UK) have conducted several biomineralization experiments in simulated porous media reactors. Microscopy has proven to be one of the most useful analytical tools in these studies, providing the ability to non-invasively visualize, differentiate, and quantify the various components, including the cells, cell matrix, and mineral precipitates. Because of the possibility of real-time observation and the lack of dehydration artifacts, microscopy has been tremendously useful for elucidating the temporal and spatial relationships of these components.
Internally coated endotracheal tubes with silver sulfadiazine in polyurethane to prevent bacterial colonization: A clinical trial
(2008-04) Berra, Lorenzo; Kolobow, Theodor; Laquerriere, Patrice; Pitts, Betsey; Bramati, Simone; Pohlmann, Joshua; Marelli, Chiara; Panzeri, Miriam; Brambillasca, Pietro; Villa, Federica; Baccarelli, Andrea; Bouthors, Sylvie; Stelfox, Henry T.; Bigatello, Luca M.; Moss, Joel; Pesenti, Antonio
OBJECTIVE: Coated medical devices have been shown to reduce catheter-related infections. We coated endotracheal tubes (ETT) with silver sulfadiazine (SSD), and tested them in a clinical study to assess the feasibility, safety, and efficacy of preventing bacterial colonization. DESIGN: A prospective, randomized clinical trial, phase I-II. SETTING: Academic intensive care unit (ICU). PARTICIPANTS: Forty-six adult patients expected to need 12-24 h of intubation were randomized into two groups. INTERVENTIONS: Patients were randomized to be intubated with a standard non-coated ETT (St-ETT, n = 23; control group), or with a SSD-coated ETT (SSD-ETT, n = 23). MEASUREMENTS AND RESULTS: Coating with SSD prevented bacterial colonization of the ETT (frequency of colonization: SSD-ETT 0/23, St-ETT 8/23; p < 0.01). No organized bacterial biofilm could be identified on the lumen of any ETT; however, SSD was associated with a thinner mucus layer (in the SSD-ETT secretion deposits ranged from 0 to 200 µm; in the St-ETT deposits ranged between 50 and 700 µm). No difference was observed between the two groups in the tracheobronchial brush samples (frequency of colonization: SSD-ETT 0/23, St-ETT 2/23; p = 0.48). No adverse reactions were observed with the implementation of the novel device. CONCLUSION: SSD-ETT can be safely used in preventing bacterial colonization and narrowing of the ETT in patients intubated for up to 24 h (mean intubation time 16h).
Localized gene expression in Pseudomonas aeruginosa biofilms
(2008-05) Lenz, Ailyn P.; Williamson, Kerry S.; Pitts, Betsey; Stewart, Philip S.; Franklin, Michael J.
Gene expression in biofilms is dependent on bacterial responses to the local environmental conditions. Most techniques for studying bacterial gene expression in biofilms characterize average values over the entire population. Here, we describe the use of laser capture microdissection microscopy (LCMM) combined with multiplex quantitative real time reverse transcriptase polymerase chain reaction (qRT-PCR) to isolate and quantify RNA transcripts from small groups of cells at spatially resolved sites within biofilms. The approach was first tested and analytical parameters determined for Pseudomonas aeruginosa containing an IPTG-inducible gene for the green fluorescent protein (gfp). The results show that amounts of gfp mRNA were greatest in the top zones of the biofilms, and that gfp mRNA levels correlated with the zone of active GFP-fluorescence. The method was then used to quantify transcripts from wild-type P. aeruginosa biofilms for a housekeeping gene, acpP; the 16S rRNA; and two genes regulated by quorum-sensing, phzA1 and aprA. The results demonstrated that the amount of acpP mRNA was greatest in the top 30 microm of the biofilm, with little or no mRNA for this gene at the base of the biofilms. In contrast, 16S rRNA amounts were relatively uniform throughout biofilm strata. Using this strategy, the RNA amounts of individual genes are determined, and therefore results are dependent on both gene expression and the half-life of transcripts. Therefore, the uniform amount of rRNA throughout the biofilms is likely due to the stability of the rRNA within ribosomes. Levels of aprA mRNA showed stratification, with the greatest amounts in the upper 30 microm zone of these biofilms. The results demonstrate that mRNA levels for individual genes are not uniformly distributed throughout biofilms, but may vary by orders of magnitude over small distances. The LCMM/qRT-PCR technique can be used to resolve and quantify this RNA variability at high spatial resolution.