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Item Contributions of antibiotic penetration, oxygen limitation, and low metabolic activity to tolerance of Pseudomonas aeruginosa biofilms to ciprofloxacin and tobramycin(2003-01) Walters, Marshall C., III; Roe, Frank L.; Bugnicourt, Amandine; Franklin, Michael J.; Stewart, Philip S.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.Item Stratified growth in Pseudomonas aeruginosa biofilms(2004-10) Werner, Erin M.; Roe, Frank L.; Bugnicourt, Amandine; Franklin, Michael J.; Heydorn, Arne; Molin, Søren; Pitts, Betsey; Stewart, Philip S.In this study, stratified patterns of protein synthesis and growth were demonstrated in Pseudomonas aeruginosa biofilms. Spatial patterns of protein synthetic activity inside biofilms were characterized by the use of two green fluorescent protein (GFP) reporter gene constructs. One construct carried an isopropyl-ß-D-thiogalactopyranoside (IPTG)-inducible gfpmut2 gene encoding a stable GFP. The second construct carried a GFP derivative, gfp-AGA, encoding an unstable GFP under the control of the growth-rate-dependent rrnBp1 promoter. Both GFP reporters indicated that active protein synthesis was restricted to a narrow band in the part of the biofilm adjacent to the source of oxygen. The zone of active GFP expression was approximately 60 µm wide in colony biofilms and 30 µm wide in flow cell biofilms. The region of the biofilm in which cells were capable of elongation was mapped by treating colony biofilms with carbenicillin, which blocks cell division, and then measuring individual cell lengths by transmission electron microscopy. Cell elongation was localized at the air interface of the biofilm. The heterogeneous anabolic patterns measured inside these biofilms were likely a result of oxygen limitation in the biofilm. Oxygen microelectrode measurements showed that oxygen only penetrated approximately 50 µm into the biofilm. P. aeruginosa was incapable of anaerobic growth in the medium used for this investigation. These results show that while mature P. aeruginosa biofilms contain active, growing cells, they can also harbor large numbers of cells that are inactive and not growing.Item Physiological heterogeneity in biofilms(2008-03) Stewart, Philip S.; Franklin, Michael J.Biofilms contain bacterial cells that are in a wide range of physiological states. Within a biofilm population, cells with diverse genotypes and phenotypes that express distinct metabolic pathways, stress responses and other specific biological activities are juxtaposed. The mechanisms that contribute to this genetic and physiological heterogeneity include microscale chemical gradients, adaptation to local environmental conditions, stochastic gene expression and the genotypic variation that occurs through mutation and selection. Here, we discuss the processes that generate chemical gradients in biofilms, the genetic and physiological responses of the bacteria as they adapt to these gradients and the techniques that can be used to visualize and measure the microscale physiological heterogeneities of bacteria in biofilms.Item 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.Item Tolerance of dormant and active cells in Pseudomonas aeruginosa PA01 biofilm to antimicrobial agents(2008-10) Kim, Jaeeun; Hahn, Ji-Sook; Franklin, Michael J.; Stewart, Philip S.; Yoon, JeyongObjectives: The aim of the study was to determine the susceptibility of active and dormant cell populations from Pseudomonas aeruginosa biofilms to non-antibiotic antimicrobial agents such as chlorine, hydrogen peroxide and silver ions in comparison with antibiotics. Methods: Active cells in colony biofilm were differentially labelled by induction of a green fluorescent protein (GFP). Active and dormant cells were sorted in phosphate buffered solution by flow cytometry. Reductions in viability were determined with plate counts. Results: The spatial pattern of metabolic activity in colony biofilm was verified, and the active and dormant cells were successfully sorted according to the GFP intensity. Active cells had bigger cell size and higher intracellular density than dormant cells. While dormant cells were more tolerant to tobramycin and silver ions, active cells were more tolerant to chlorine. Metabolically active cells contain denser intracellular components that can react with highly reactive oxidants such as chlorine, thereby reducing the available concentrations of chlorine. In contrast, the concentrations of silver ions and hydrogen peroxide were constant during treatment. Aerobically grown stationary cells were significantly more tolerant to chlorine unlike other antimicrobial agents. Conclusions: Chlorine was more effective in inactivation of metabolically inactive dormant cells and also more effective under anaerobic conditions. The high oxidative reactivity and rapid decay of chlorine might influence the different antimicrobial actions of chlorine compared with antibiotics. This study contributes to understanding the effects of dormancy and the presence of oxygen on the susceptibility of P. aeruginosa biofilm to a wide range of antimicrobial agents.Item Role of antibiotic penetration limitation in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin(2000) Anderl, Jeff N.; Franklin, Michael J.; Stewart, Philip S.The penetration of two antibiotics—ampicillin and ciprofloxacin—through biofilms developed in an in vitro model system was investigated. The susceptibilities of biofilms and corresponding freely suspended bacteria, to killing by the antibiotics were also measured. Biofilms of Klebsiella pneumoniae were developed on microporous membranes resting on agar nutrient medium. The susceptibilities of planktonic cultures and biofilms to 10 times the MIC were determined. Antibiotic penetration through biofilms was measured by assaying the concentration of antibiotic that diffused through the biofilm to an overlying filter disk. Parallel experiments were performed with a mutant K. pneumoniae strain in which B-lactamase activity was eliminated. For wild-type K. pneumoniae grown in suspension culture, ampicillin and ciprofloxacin MICs were 500 and 0.18 ug/ml, respectively. The log reductions in the number of CFU of planktonic wild-type bacteria after 4 h of treatment at 10 times the MIC were 4.43 + 0.33 and 4.14 + 0.33 for ampicillin and ciprofloxacin, respectively. Biofilms of the same strain were much less susceptible, yielding log reductions in the number of CFU of – 0.06 + 0.06 and 1.02 + 0.04 for ampicillin and ciprofloxacin, respectively, for the same treatment. The number of CFU in the biofilms after 24 h of antibiotic exposure was not statistically different from the number after 4 h of treatment. Ampicillin did not penetrate wild-type K. pneumoniae biofilms, whereas ciprofloxacin and a nonreactive tracer (chloride ion) penetrated the biofilms quickly. The concentration of ciprofloxacin reached the MIC throughout the biofilm within 20 min. Ampicillin penetrated biofilms formed by a B-lactamase-deficient mutant. However, the biofilms formed by this mutant were resistant to ampicillin treatment, exhibiting a 0.18 + 0.07 log reduction in the number of CFU after 4 h of exposure and a 1.64 + 0.33 log reduction in the number of CFU after 24 h of exposure. Poor penetration contributed to wild-type biofilm resistance to ampicillin but not to ciprofloxacin. The increased resistance of the wild-type strain to ciprofloxacin and the mutant strain to ampicillin and ciprofloxacin could not be accounted for by antibiotic inactivation or slow diffusion since these antibiotics fully penetrated the biofilms. These results suggest that some other resistance mechanism is involved for both agents.Item A repeatable laboratory method for testing the efficacy of biocides against toilet bowl biofilms(2001-07) Pitts, Betsey; Willse, Alan Ray; McFeters, Gordon A.; Hamilton, Martin A.; Zelver, Nick; Stewart, Philip S.Aims: The purpose of this study was to develop a laboratory biofilm growth reactor system that simulated the toilet bowl environment and which could be used for biocide efficacy testing. Methods and Results: A microbial biofilm reactor system incorporating intermittent flow and nutrient provision was designed. The reactor system was open to the air and was inoculated with organisms collected from toilet bowl biofilms. Once per hour, reactors were supplied with a nutrient solution for a period of 5 min, then flushed and refilled with tap water or tap water amended with chlorine. Quantitative measures of the rate and extent of biofilm accumulation were defined. Biofilm accumulated in untreated reactors to cell densities of 108 cfu cm–2 after approximately 1 week. Biofilm accumulation was also observed in reactors in the continuous presence of several milligrams per litre of free chlorine. Repeatability standard deviations for the selected efficacy measures were low, indicating high repeatability between experiments. Log reduction values of viable cell numbers were within ranges observed with standard suspension and hard surface disinfection tests. Biofilm accumulated in laboratory reactors approximately seven times faster than it did in actual toilet bowls. The same ranking was achieved in tests between laboratory biofilms and field-grown biofilms with three of the four measures, using three different concentrations of chlorine. Conclusions: This reactor system has been shown to simulate, in a repeatable way, the accumulation of bacterial biofilm that occurs in toilet bowls. The results demonstrate that this system can provide repeatable assays of the efficacy of chlorine against those biofilms. Significance and Impact of the Study: The laboratory biofilm reactor system described herein can be used to evaluate potential antimicrobial and antifouling treatments for control of biofilm formation in toilet bowls.Item Assessing biofouling on polyamide reverse osmosis (RO) membrane surfaces in a laboratory system(2010-04) Khan, Mohiuddin M. T.; Stewart, Philip S.; Moll, D. J.; Mickols, W. E.; Burr, Mark D.; Nelson, Sara E.; Camper, Anne K.Biofouling of reverse osmosis (RO) membranes is a major impediment in both wastewater reuse and desalination of sea/brackish waters. A benefit to the industry would be a simple screening approach to evaluate biofouling resistant RO membranes for their propensity to biofoulants. To observe the relationship between initial membrane productivity and control of biofilm formation governed by surface modification to the aromatic polyamide thin-film composite RO membranes, three different RO membranes developed by the FilmTec Corporation including FilmTec’s commercial membrane BW30 (RO#1) and two experimental membranes (RO #2 and #3) were used. RO #2 and RO #3 were modified with a proprietary aliphatic group and with an extra proprietary aromatic group, respectively. Membrane swatches were fixed on coupons in rotating disk reactor systems without filtration and exposed to water with indigenous organisms supplemented with 1.5 mg/L organic carbon under continuous flow. After biofouling had developed, the membranes were sacrificed and subjected to several analyses. Staining and epifluorescence microscopy revealed more cells on RO #2 and #3 compared to RO #1. Based on image analysis of 5-µmthick stained biofoulant cryo-sections, the accumulation of hydrated biofoulants on RO #1 and #3 were from 0.87 to 1.26µm/day, which was lower than that on RO#2 (2.19µm/day). Biofoulants increased the hydrophobicity of RO #2 to the greatest amount, up to 32°, as determined by contact angle. In addition, a wide range of changes of the chemical elements of the RO surfaces was observed with X-ray photoelectron spectroscopy analysis. RO #2 with the highest initial membrane productivity showed the poorest biofouling resistance. A combination of these novel approaches showed good agreement and suggested that membrane productivity, heterogeneity of anti-biofouling agents on membrane surface, stability of surface chemical elements and the role of virgin RO surface hydrophobicity should be jointly considered during the development of anti-biofouling polyamide thin-film RO surfaces.Item Robustness analysis of culturing perturbations on Escherichia coli colony biofilm beta-lactam and aminoglycoside antibiotic tolerance(2010-07) Zuroff, Trevor R.; Bernstein, Hans C.; Lloyd-Randolfi, J.; Jimenez-Taracido, L.; Stewart, Philip S.; Carlson, Ross P.BACKGROUND: Biofilms are ubiquitous. For instance, the majority of medical infections are thought to involve biofilms. However even after decades of investigation, the in vivo efficacy of many antimicrobial strategies is still debated, suggesting there is a need for better understanding of biofilm antimicrobial tolerances. The current study's goal is to characterize the robustness of biofilm antibiotic tolerance to medically and industrially relevant culturing perturbations. By definition, robust systems will return similar, predictable responses when perturbed, while non-robust systems will return very different, and potentially unpredictable, responses. The predictability of an antibiotic tolerance response is essential to developing, testing, and employing antimicrobial strategies. RESULTS: The antibiotic tolerance of Escherichia coli colony biofilms was tested against beta-lactam and aminoglycoside class antibiotics. Control scenario tolerances were compared to tolerances under culturing perturbations including 1) different nutritional environments 2) different temperatures 3) interruption of cellular quorum sensing and 4) different biofilm culture ages. Here, antibiotic tolerance was defined in terms of culturable biofilm cells recovered after a twenty four hour antibiotic treatment.Colony biofilm antibiotic tolerances were not robust to perturbations. Altering basic culturing parameters like nutritional environment or temperature resulted in very different, non-intuitive antibiotic tolerance responses. Some minor perturbations—like increasing the glucose concentration from 0.1 to 1 g/L—caused a ten-million fold difference in culturable cells over a twenty four hour antibiotic treatment. CONCLUSIONS: The current study presents a basis for robustness analysis of biofilm antibiotic tolerance. Biofilm antibiotic tolerance can vary in unpredictable manners based on modest changes in culturing conditions. Common antimicrobial testing methods, which only consider a single culturing condition, are not desirable since slight culturing variations can lead to very different outcomes. The presented data suggest it is essential to test antimicrobial strategies over a range of culturing perturbations relevant to the targeted application. In addition, the highly dynamic antibiotic tolerance responses observed here may explain why some current antimicrobial strategies occasionally fail.Item Spatial and temporal patterns of biocide action against Staphylococcus epidermidis biofilms(2010-05) Davison, William M.; Pitts, Betsey; Stewart, Philip S.The dynamic antimicrobial action of chlorine, a quaternary ammonium compound, glutaraldehyde, and nisin within biofilm cell clusters of Staphylococcus epidermidis was investigated using time-lapse confocal scanning laser microscopy. The technique allowed for the simultaneous imaging of changes in biofilm structure and disruption of cellular membrane integrity through the loss of an unbound fluorophore loaded into bacterial cells prior to antimicrobial challenge. Each of the four antimicrobial agents produced distinct spatial and temporal patterns of fluorescence loss. The antimicrobial action of chlorine was localized around the periphery of biofilm cell clusters. Chlorine was the only antimicrobial agent that caused any biofilm removal. Treatment with the quaternary ammonium compound caused membrane permeabilization that started at the periphery of cell clusters, then migrated steadily inward. A secondary pattern superimposed on the penetration dynamic suggested a subpopulation of less-susceptible cells. These bacteria lost fluorescence much more slowly than the majority of the population. Nisin caused a rapid and uniform loss of green fluorescence from all parts of the biofilm without any removal of biofilm. Glutaraldehyde caused no biofilm removal and also no loss of membrane integrity. Measurements of biocide penetration and action time at the center of cell clusters yielded 46 min for 10 mg liter-1 chlorine, 21 min for 50 mg liter-1 chlorine, 25 min for the quaternary ammonium compound, and 4 min for nisin. These results underscore the distinction between biofilm removal and killing and reinforce the critical role of biocide reactivity in determining the rate of biofilm penetration.