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Biomedical subjects

Anne K Camper

Publications and source records attributed to Anne K Camper.

14 recordsLinked to original sources
PubMed2006

Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells.

The differentiation between live and dead bacterial cells presents an important challenge in many microbiological applications. Due to the persistence of DNA in the environment after cells have lost viability, DNA-based detection methods cannot differentiate whether positive signals originate from live or dead bacterial targets. We present here a novel chemical, propidium monoazide (PMA), that (like propidium iodide) is highly selective in penetrating only into 'dead' bacterial cells with compromised membrane integrity but not into live cells with intact cell membranes/cell walls. Upon intercalation in the DNA of dead cells, the photo-inducible azide group allows PMA to be covalently cross-linked by exposure to bright light. This process renders the DNA insoluble and results in its loss during subsequent genomic DNA extraction. Subjecting a bacterial population comprised of both live and dead cells to PMA treatment thus results in selective removal of DNA from dead cells. We provide evidence that this chemical can be applied to a wide range of species across the bacterial kingdom presenting a major advantage over ethidium monoazide (EMA). The general application of EMA is hampered by the fact that the chemical can also penetrate live cells of some bacterial species. Transport pumps actively export EMA out of metabolically active cells, but the remaining EMA level can lead to substantial loss of DNA. The higher charge of PMA might be the reason for the higher impermeability through intact cell membranes, thus avoiding DNA loss.

Azides↗
PubMed2006

Effect of substrate concentration on dual-species biofilm population densities of Klebsiella oxytoca and Burkholderia cepacia in porous media.

The long-term operation of bioremediation technologies relies on the success of the contaminant-degrading microorganism(s) to compete for available resources with microorganisms already present in an aquifer or those that may contaminate a bioreactor. Though research has been performed studying the interaction of multiple species in batch and chemostat reactors, little work has been done looking at multi-species interactions in environments that more closely resemble field-scale applications. The research presented herein examined the interaction of Burkholderia cepacia PR1-pTOM(31c), an aerobic trichloroethylene (TCE)-degrading bacterium, with Klebsiella oxytoca, a facultative bacterium, in a flow-through porous media (PM) reactor. Growth characteristics and population distributions in PM were compared to previously reported values from batch and chemostat reactors. The faster growing organism in batch experiments (K. oxytoca) did not always have the greater population density in dual-species PM experiments. The biofilm population distribution was influenced by substrate concentration, with B. cepacia having a greater dual-species population density than K. oxytoca at a low (30 mg/L dissolved organic carbon [DOC]) substrate concentration and K. oxytoca having a greater population density at a high (700 mg/L DOC) substrate concentration. This change in species population distribution with change in substrate concentration, which was not observed in batch reactors, was also observed in chemostat reactors. Therefore, manipulation of substrate concentration enabled the control of species dominance to the advantage of the TCE degrading population in this dual-species PM system and may provide a mechanism to enhance bioremediation scenarios involving TCE or other contaminants of concern.

Biodegradation, Environmental↗
PubMed2006

Selective removal of DNA from dead cells of mixed bacterial communities by use of ethidium monoazide.

The distinction between viable and dead bacterial cells poses a major challenge in microbial diagnostics. Due to the persistence of DNA in the environment after cells have lost viability, DNA-based quantification methods overestimate the number of viable cells in mixed populations or even lead to false-positive results in the absence of viable cells. On the other hand, RNA-based diagnostic methods, which circumvent this problem, are technically demanding and suffer from some drawbacks. A promising and easy-to-use alternative utilizing the DNA-intercalating dye ethidium monoazide bromide (EMA) was published recently. This chemical is known to penetrate only into "dead" cells with compromised cell membrane integrity. Subsequent photoinduced cross-linking was reported to inhibit PCR amplification of DNA from dead cells. We provide evidence here that in addition to inhibition of amplification, most of the DNA from dead cells is actually lost during the DNA extraction procedure, probably together with cell debris which goes into the pellet fraction. Exposure of bacteria to increasing stress and higher proportions of dead cells in defined populations led to increasing loss of genomic DNA. Experiments were performed using Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium as model pathogens and using real-time PCR for their quantification. Results showed that EMA treatment of mixed populations of these two species provides a valuable tool for selective removal of DNA of nonviable cells by using conventional extraction protocols. Furthermore, we provide evidence that prior to denaturing gradient gel electrophoresis, EMA treatment of a mature mixed-population drinking-water biofilm containing a substantial proportion of dead cells can result in community fingerprints dramatically different from those for an untreated biofilm. The interpretation of such fingerprints can have important implications in the field of microbial ecology.

Affinity Labels↗
PubMed2006

Identification of Staphylococcus aureus proteins recognized by the antibody-mediated immune response to a biofilm infection.

Staphylococcus aureus causes persistent, recurrent infections (e.g., osteomyelitis) by forming biofilms. To survey the antibody-mediated immune response and identify those proteins that are immunogenic in an S. aureus biofilm infection, the tibias of rabbits were infected with methicillin-resistant S. aureus to produce chronic osteomyelitis. Sera were collected prior to infection and at 14, 28, and 42 days postinfection. The sera were used to perform Western blot assays on total protein from biofilm grown in vitro and separated by two-dimensional gel electrophoresis. Those proteins recognized by host antibodies in the harvested sera were identified via matrix-assisted laser desorption ionization-time of flight analysis. Using protein from mechanically disrupted total and fractionated biofilm protein samples, we identified 26 and 22 immunogens, respectively. These included a cell surface-associated beta-lactamase, lipoprotein, lipase, autolysin, and an ABC transporter lipoprotein. Studies were also performed using microarray analyses and confirmed the biofilm-specific up-regulation of most of these genes. Therefore, although the biofilm antigens are recognized by the immune system, the biofilm infection can persist. However, these proteins, when delivered as vaccines, may be important in directing the immune system toward an early and effective antibody-mediated response to prevent chronic S. aureus infections. Previous works have identified S. aureus proteins that are immunogenic during acute infections, such as sepsis. However, this is the first work to identify these immunogens during chronic S. aureus biofilm infections and to simultaneously show the global relationship between the antigens expressed during an in vivo infection and the corresponding in vitro transcriptomic and proteomic gene expression levels.

Animals↗
PubMed2005

Assessment of the ability of the bioelectric effect to eliminate mixed-species biofilms.

Microbes have been able to persist in water distribution systems through the development of multicellular communities known as biofilms. This study evaluated the usefulness of the bioelectric effect for the elimination of water distribution system biofilms from annular reactors. The bioelectric effect did not have any bactericidal action either alone or when coupled with free chlorine.

Bioelectric Energy Sources↗
PubMed2004

Involvement of humic substances in regrowth.

There appear to be interactions in the distribution system that complicate the ability to use AOC/BDOC as an independent assessment of regrowth potential. Two such complications are the limitation of the assays themselves and the potential interaction between the organic carbon concentration with the presence of disinfectants and pipe materials. To address these interactions, a series of experiments spanning several years have been conducted in model distribution systems at the Center for Biofilm Engineering (CBE) using soil-derived humics. When compared to easily utilized organics, humic substances supported the same order of magnitude of biofilm organisms. As carbon concentration was increased from 500 to 1000 to 2000 ppb, there was no increase in growth rate of the organisms, suggesting zero-order kinetics. If the system was chlorinated, there was less biomass, but growth rates were higher. In the presence of corrosion products, humic-fed systems supported more organisms than a control system fed biologically treated water. When free chlorine was maintained at a residual of about 0.2 mg/l, biofilm numbers on the surfaces were reduced. Phosphate alone did not result in fewer bacteria, while a combination of chorine and phosphate had the best results (lowest biofilm numbers). Adjustment to pH 9 was not effective. Recently completed work compared increasing levels of humic substances in the presence of free chlorine and monochloramine on biofilm growth on a number of surfaces (PVC, epoxy, cement, ductile iron). As the concentration of humic substances was increased from 0, 0.5 to 2 mg/l, there was an increase in biofilm numbers on all surfaces. This effect was the most pronounced on iron surfaces. These results illustrate that carbon compounds not measured by the BDOC or AOC tests may profoundly influence biofilm numbers. In addition, iron surfaces are at much higher risk for elevated biofilm counts in the presence of humic substances, even if disinfection is practiced. However, corrosion control may mitigate this interaction.

Bacteria↗
PubMed2004

Development of a toolbox to assess microbial contamination risks in small water systems.

Individual and small water systems account for the majority of waterborne disease outbreaks recorded in the United States each year. To address this problem a project was undertaken to develop a comprehensive self-assessment toolbox that could be used by small water system personnel to determine where their system has the greatest potential risks from microbial contamination. The toolbox components consist of: (1) a survey that asks specific questions; (2) a ranking tool that computes numerical scores for water system components based on survey answers; (3) comments and results from the ranking tool; (4) a guidance document to help the user understand why certain conditions may represent a risk; and (5) instructions for using the toolbox. A unique feature of the ranking tool is the ability to input expert opinion in the form of scores for each answer and weighting factors. Weighting factors are derived using ranked, pairwise comparisons, and then used to determine numerical scores for system components. Toolbox administrators are allowed to modify weighting factors used by the ranking tool application, thus allowing input of expert opinion.

Disease Outbreaks↗
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↗
PubMed2002

Minimizing biofilm in the presence of iron oxides and humic substances.

Based upon circumstantial evidence linking elevated coliform bacteria counts in drinking water distribution systems with unlined cast iron pipe, it was hypothesized that adsorption of humic substances by iron oxide containing corrosion products (CPs) can stimulate and/or support biofilm development. Using porous media consisting of iron-oxide-coated glass beads (IOCBs) or actual iron CPs, experiments were performed to evaluate the effectiveness of different corrosion control and disinfection treatments in reducing biofilm when humic substances were the carbon source. Free chlorine was the most effective treatment in minimizing biofilm. Addition of phosphate alone did not significantly reduce biofilm using the CPs, but there was weak evidence it did using the IOCBs. The combination of free chlorine and phosphate was more effective at minimizing biofilm than free chlorine alone when CPs were the media. The presence of humic substances was a major factor when considering biofilm minimization based on results of experiments using both types of iron oxide media. The combination of humic substances and CPs led to an increase in biofilm biomass when free chlorine was not present, similar to conditions that could occur at distribution system dead-ends. Treatment to raise the pH to 9 did not reduce biofilm in experiments using both media, and actually increased biofilm in the experiment using CPs under the conditions tested.

Bacteria↗
PubMed2002

Chlorination of model drinking water biofilm: implications for growth and organic carbon removal.

The influence of chlorine on biofilm in low organic carbon environments typical of drinking water or industrial process water was examined by comparing biomass and kinetic parameters for biofilm growth in a chlorinated reactor to those in a non-chlorinated control. Mixed-population heterotrophic biofilms were developed in rotating annular reactors under low concentration, carbon-limited conditions (< 2 mg/L as carbon) using three substrate groups (amino acids, carbohydrates and humic substances). Reactors were operated in parallel under identical conditions with the exception that chlorine was added to one reactor at a dose sufficient to maintain a free chlorine residual of 0.09-0.15 mg/L in the effluent. The presence of free chlorine resulted in development of less biofilm biomass compared to the control for all substrates investigated. However, specific growth and organic carbon removal rates were on the average five times greater for chlorinated biofilm compared to the control. Observed yield values were less for chlorinated biofilm. Although chlorinated biofilm's specific organic carbon removal rate was high, the low observed yield indicated organic carbon was being utilized for purposes other than creating new cell biomass. The impacts of free chlorine on mixed-population biofilms in low-nutrient environments were different depending upon the available substrate. Biofilms grown using amino acids exhibited the least difference between control and chlorinated kinetic parameters; biofilm grown using carbohydrates had the greatest differences. These findings are particularly relevant to the fundamental kinetic parameters used in models of biofilm growth in piping systems that distribute chlorinated, low-carbon-concentration water.

Biofilms↗
PubMed2002

Modified enzyme activity assay to determine biofilm biomass.

An assay of potential exoproteolytic enzyme activity was modified to quantitatively measure the biomass of attached biofilm. The assay utilized the nonfluorescent compound L-leucine-beta-naphthylamide (LLbetaN) that becomes fluorescent when bacterial exoenzymes break the peptide bond, releasing the fluorochrome beta-naphthylamine. Fluorescence development was measured by pumping the liquid phase of a biofilm sample through a fluorescence detector and recording the detector output using a personal computer. A significant linear relationship was shown to exist between the rate of fluorescence development and the biofilm's biomass as carbon, determined using total direct cell counts, measured cell volumes and an existing relationship between cell volume and cell carbon. The technique was used to measure biofilm biomass for experiments where iron oxides were the substratum. Biofilm biomass measurements made using heterotrophic plate counts (HPCs) on R2A medium were shown to correlate well to biomass measurements made using the modified enzyme assay. The technique was shown to be sufficiently sensitive to measure biomass on samples containing little biofilm biomass, such as those exposed to free chlorine. While granular and porous media were used for the experiments presented, small biofilm coupons could easily be used to measure biofilm biomass, expanding the number of possible applications for the enzyme assay technique.

Bacteria↗
PubMed2002

Molecular interactions in biofilms.

A biofilm may be defined as a microbially derived, sessile community characterized by cells that attach to an interface, embed in a matrix of exopolysaccharide, and demonstrate an altered phenotype. This review covers the current understanding of the nature of biofilms and the impact that molecular interactions may have on biofilm development and phenotype using the motile gram-negative rod Pseudomonas aeruginosa and the nonmotile gram-positive cocci Staphylococcus aureus as examples.

Biofilms↗
PubMed2002

Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm.

Complementary approaches were employed to characterize transitional episodes in Pseudomonas aeruginosa biofilm development using direct observation and whole-cell protein analysis. Microscopy and in situ reporter gene analysis were used to directly observe changes in biofilm physiology and to act as signposts to standardize protein collection for two-dimensional electrophoretic analysis and protein identification in chemostat and continuous-culture biofilm-grown populations. Using these approaches, we characterized five stages of biofilm development: (i) reversible attachment, (ii) irreversible attachment, (iii) maturation-1, (iv) maturation-2, and (v) dispersion. Biofilm cells were shown to change regulation of motility, alginate production, and quorum sensing during the process of development. The average difference in detectable protein regulation between each of the five stages of development was 35% (approximately 525 proteins). When planktonic cells were compared with maturation-2 stage biofilm cells, more than 800 proteins were shown to have a sixfold or greater change in expression level (over 50% of the proteome). This difference was higher than when planktonic P. aeruginosa were compared with planktonic cultures of Pseudomonas putida. Las quorum sensing was shown to play no role in early biofilm development but was important in later stages. Biofilm cells in the dispersion stage were more similar to planktonic bacteria than to maturation-2 stage bacteria. These results demonstrate that P. aeruginosa displays multiple phenotypes during biofilm development and that knowledge of stage-specific physiology may be important in detecting and controlling biofilm growth.

Amino Acids↗