PubMed Health⌕ Search

Biomedical subjects

Philip S Stewart

Publications and source records attributed to Philip S Stewart.

14 recordsLinked to original sources
PubMed2004

Magnetic resonance microscopy of biofilm structure and impact on transport in a capillary bioreactor.

Microorganisms that colonize surfaces, biofilms, are of significant importance due to their role in medical infections, subsurface contaminant remediation, and industrial processing. Spatially resolved data on the distribution of biomass within a capillary bioreactor, the heterogeneity of the biofilm itself and the impact on transport dynamics for a Staphylococcus epidermidis biofilm in the natural growth state are presented. The data demonstrate the ability of magnetic resonance microscopy to study spatially resolved processes in bacterial biofilms, thus providing a basis for future studies of spatially resolved metabolism and in vivo clinical detection.

Biofilms↗
PubMed2004

Modeling antibiotic tolerance in biofilms by accounting for nutrient limitation.

A mathematical model of biofilm dynamics was used to investigate the protection from antibiotic killing that can be afforded to microorganisms in biofilms based on a mechanism of localized nutrient limitation and slow growth. The model assumed that the rate of killing by the antibiotic was directly proportional to the local growth rate. Growth rates in the biofilm were calculated by using the local concentration of a single growth-limiting substrate with Monod kinetics. The concentration profile of this metabolic substrate was calculated by solving a reaction-diffusion problem. The model predicted the following features: stratified patterns of growth with zones of no growth in the biofilm interior, slow killing of biofilm microorganisms that was further retarded as the initial biofilm thickness increased, nonuniform spatial patterns of killing inside the biofilm, biofilm killing rates that decrease in a nonlinear way as the concentration of the growth-limiting substrate feeding the biofilm is decreased, and heightened tolerance when external mass transfer resistance is manifested. This modeling study also provides motivation for further investigation of a hypothetical cell state in which damaged cells score as nonviable but continue to consume substrate. The existence of such a cell state can further retard biofilm killing, according to the simulations. The results support the important contributions of nutrient limitation and slow growth to the antibiotic tolerance of microorganisms in biofilms.

Anti-Bacterial Agents↗
PubMed2004

Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms.

The role of oxygen limitation in protecting Pseudomonas aeruginosa strains growing in biofilms from killing by antibiotics was investigated in vitro. Bacteria in mature (48-h-old) colony biofilms were poorly killed when they were exposed to tobramycin, ciprofloxacin, carbenicillin, ceftazidime, chloramphenicol, or tetracycline for 12 h. It was shown with oxygen microelectrodes that these biofilms contain large anoxic regions. Oxygen penetrated about 50 microm into the biofilms, which averaged 210 microm thick. The region of active protein synthesis was visualized by using an inducible green fluorescent protein. This zone was also limited to a narrow band, approximately 30 microm wide, adjacent to the air interface of the biofilm. The bacteria in mature biofilms exhibited a specific growth rate of only 0.02 h(-1). These results show that 48-h-old colony biofilms are physiologically heterogeneous and that most of the cells in the biofilm occupy an oxygen-limited, stationary-phase state. In contrast, bacteria in 4-h-old colony biofilms were still growing, active, and susceptible to antibiotics when they were challenged in air. When 4-h-old colony biofilms were challenged under anaerobic conditions, the level of killing by antibiotics was reduced compared to that for the controls grown aerobically. Oxygen limitation could explain 70% or more of the protection afforded to 48-h-old colony biofilms for all antibiotics tested. Nitrate amendment stimulated the growth of untreated control P. aeruginosa isolates grown under anaerobic conditions but decreased the susceptibilities of the organisms to antibiotics. Local oxygen limitation and the presence of nitrate may contribute to the reduced susceptibilities of P. aeruginosa biofilms causing infections in vivo.

Aerobiosis↗
PubMed2003

A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance.

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.

Animals↗
PubMed2003

Pretreatment for membrane water treatment systems: a laboratory study.

The goal of the work was to determine if biological treatment of water containing soil-derived humic substances has the potential for reducing the fouling of membranes used in water treatment. Laboratory scale biological filters containing biologically active carbon or iron oxide coated sand were fed humic-laden water with or without prechlorination. This stream was split, with half being further treated by microfiltration. Treated water was assessed for total organic carbon removal and biofouling potential using a glass bead assay and membrane assay for total cell counts, fouling layer thickness, and flux reduction. A combination of these assays provided more insight than any single measurement. Compared to untreated control water, biological treatment was capable of reducing downstream fouling of membrane systems. For example, fouling layer thickness was reduced by half after biological treatment, and cell counts were reduced four- to five-fold. Biological treatment coupled with microfiltration provided the best reduction of fouling, while prechlorination did not appear to impact the process. These results suggest that biological treatment may be valuable in reducing membrane fouling while reducing the amount of disinfectants used in pretreatment.

Biofilms↗
PubMed2003

A microtiter-plate screening method for biofilm disinfection and removal.

A quantitative spectrophotometric method was developed to measure the removal and killing efficacy of antibiofilm agents. Biofilms of Pseudomonas aeruginosa or Staphylococcus epidermidis were grown in 96-well plates, treated with an agent, then stained with either the biomass indicator crystal violet or the respiratory indicator 5-cyano-2,3-ditolyl tetrazolium chloride. This rapid screening method is sensitive enough to elucidate concentration-response relationships as well as differences between species responses to treatments. Using these assays, agents can be ranked by their ability to remove or kill biofilm.

Biofilms↗
PubMed2003

Contributions of antibiotic penetration, oxygen limitation, and low metabolic activity to tolerance of Pseudomonas aeruginosa biofilms to ciprofloxacin and tobramycin.

The roles of slow antibiotic penetration, oxygen limitation, and low metabolic activity in the tolerance of Pseudomonas aeruginosa in biofilms to killing by antibiotics were investigated in vitro. Tobramycin and ciprofloxacin penetrated biofilms but failed to effectively kill the bacteria. Bacteria in colony biofilms survived prolonged exposure to either 10 micro g of tobramycin ml(-1)or 1.0 micro g of ciprofloxacin ml(-1). After 100 h of antibiotic treatment, during which the colony biofilms were transferred to fresh antibiotic-containing plates every 24 h, the log reduction in viable cell numbers was only 0.49 +/- 0.18 for tobramycin and 1.42 +/- 0.03 for ciprofloxacin. Antibiotic permeation through colony biofilms, indicated by a diffusion cell bioassay, demonstrated that there was no acceleration in bacterial killing once the antibiotics penetrated the biofilms. These results suggested that limited antibiotic diffusion is not the primary protective mechanism for these biofilms. Transmission electron microscopic observations of antibiotic-affected cells showed lysed, vacuolated, and elongated cells exclusively near the air interface in antibiotic-treated biofilms, suggesting a role for oxygen limitation in protecting biofilm bacteria from antibiotics. To test this hypothesis, a microelectrode analysis was performed. The results demonstrated that oxygen penetrated 50 to 90 micro m into the biofilm from the air interface. This oxic zone correlated to the region of the biofilm where an inducible green fluorescent protein was expressed, indicating that this was the active zone of bacterial metabolic activity. These results show that oxygen limitation and low metabolic activity in the interior of the biofilm, not poor antibiotic penetration, are correlated with antibiotic tolerance of this P. aeruginosa biofilm system.

Biofilms↗
PubMed2003

Role of nutrient limitation and stationary-phase existence in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin.

Biofilms formed by Klebsiella pneumoniae resisted killing during prolonged exposure to ampicillin or ciprofloxacin even though these agents have been shown to penetrate bacterial aggregates. Bacteria dispersed from biofilms into medium quickly regained most of their susceptibility. Experiments with free-floating bacteria showed that stationary-phase bacteria were protected from killing by either antibiotic, especially when the test was performed in medium lacking carbon and nitrogen sources. These results suggested that the antibiotic tolerance of biofilm bacteria could be explained by nutrient limitation in the biofilm leading to stationary-phase existence of at least some of the cells in the biofilm. This mechanism was supported by experimental characterization of nutrient availability and growth status in biofilms. The average specific growth rate of bacteria in biofilms was only 0.032 h(-1) compared to the specific growth rate of planktonic bacteria of 0.59 h(-1) measured in the same medium. Glucose did not penetrate all the way through the biofilm, and oxygen was shown to penetrate only into the upper 100 micro m. The specific catalase activity was elevated in biofilm bacteria to a level similar to that of stationary-phase planktonic cells. Transmission electron microscopy revealed that bacteria were affected by ampicillin near the periphery of the biofilm but were not affected in the interior. Taken together, these results indicate that K. pneumoniae in this system experience nutrient limitation locally within the biofilm, leading to zones in which the bacteria enter stationary phase and are growing slowly or not at all. In these inactive regions, bacteria are less susceptible to killing by antibiotics.

Ampicillin↗
PubMed2002

Mechanisms of antibiotic resistance in bacterial biofilms.

Bacteria that attach to a surface and grow as a biofilm are protected from killing by antibiotics. Reduced antibiotic susceptibility contributes to the persistence of biofilm infections such as those associated with implanted devices. The protective mechanisms at work in biofilms appear to be distinct from those that are responsible for conventional antibiotic resistance. In biofilms, poor antibiotic penetration, nutrient limitation and slow growth, adaptive stress responses, and formation of persister cells are hypothesized to constitute a multi-layered defense. The genetic and biochemical details of these biofilm defenses are only now beginning to emerge. Each gene and gene product contributing to this resistance may be a target for the development of new chemotherapeutic agents. Disabling biofilm resistance may enhance the ability of existing antibiotics to clear infections involving biofilms that are refractory to current treatments.

Bacterial Adhesion↗
PubMed2002

Penetration of rifampin through Staphylococcus epidermidis biofilms.

Rifampin penetrated biofilms formed by Staphylococcus epidermidis but failed to effectively kill the bacteria. Penetration was demonstrated by a simple diffusion cell bioassay and by transmission electron microscopic observation of antibiotic-affected cells at the distal edge of the biofilm.

Antibiotics, Antitubercular↗
PubMed2002

Transmission electron microscopic study of antibiotic action on Klebsiella pneumoniae biofilm.

The penetration of ampicillin and ciprofloxacin through biofilms formed by Klebsiella pneumoniae was confirmed by transmission electron microscopic observation of antibiotic-affected cells at the distal edge of the biofilm. Because the bacteria nevertheless survived antibiotic treatment, some protective mechanism other than inadequate penetration must have been at work in the biofilm.

Ampicillin↗