Scholarly Work - Center for Biofilm Engineering
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/9335
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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 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 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 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.Item 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.Item 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 Δrmf 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.Item 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.Item Study of the effect of antimicrobial peptide mimic, CSA-13, on an established biofilm formed by Pseudomonas aeruginosa(2013-04) Nagant, C.; Pitts, Betsey; Stewart, Philip S.; Feng, Y.; Savage, P. B.; Dehaye, J. P.The formation of a Pseudomonas aeruginosa biofilm, a complex structure enclosing bacterial cells in an extracellular polymeric matrix, is responsible for persistent infections in cystic fibrosis patients leading to a high rate of morbidity and mortality. The protective environment created by the tridimensional structure reduces the susceptibility of the bacteria to conventional antibiotherapy. Cationic steroid antibiotics (CSA)-13, a nonpeptide mimic of antimicrobial peptides with antibacterial activity on planktonic cultures, was evaluated for its ability to interact with sessile cells. Using confocal laser scanning microscopy, we demonstrated that the drug damaged bacteria within an established biofilm showing that penetration did not limit the activity of this antimicrobial agent against a biofilm. When biofilms were grown during exposure to shear forces and to a continuous medium flow allowing the development of robust structures with a complex architecture, CSA-13 reached the bacteria entrapped in the biofilm within 30 min. The permeabilizing effect of CSA-13 could be associated with the death of the bacteria. In static conditions, the compound did not perturb the architecture of the biofilm. This study confirms the potential of CSA-13 as a new strategy to combat persistent infections involving biofilms formed by P. aeruginosa.Item 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.Item 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.