Mary Jo Kirisits

Pseudomonas aeruginosa Rugose Small-Colony Variants Have Adaptations That Likely Promote Persistence in the Cystic Fibrosis Lung

Pseudomonas aeruginosa is recognized for its ability to colonize diverse habitats, ranging from soil to immunocompromised people. The formation of surface-associated communities called biofilms is one factor thought to enhance colonization and persistence in these diverse environments. Another factor is the ability of P. aeruginosa to diversify genetically, generating phenotypically distinct subpopulations. One manifestation of diversification is the appearance of colony morphology variants on solid medium. Both laboratory biofilm growth and chronic cystic fibrosis (CF) airway infections produce rugose small-colony variants (RSCVs) characterized by wrinkled, small colonies and an elevated capacity to form biofilms. Previous reports vary on the characteristics attributable to RSCVs. Here we report a detailed comparison of clonally related wild-type and RSCV strains isolated from both CF sputum and laboratory biofilm cultures. The clinical RSCV had many characteristics in common with biofilm RSCVs. Transcriptional profiling and Biolog phenotypic analysis revealed that RSCVs display increased expression of the pel and psl polysaccharide gene clusters, decreased expression of motility functions, and a defect in growth on some amino acid and tricarboxylic acid cycle intermediates as sole carbon sources. RSCVs also elicited a reduced chemokine response from polarized airway epithelium cells compared to wild-type strains. A common feature of all RSCVs analyzed in this study is increased levels of the intracellular signaling molecule cyclic di-GMP (c-di-GMP). To assess the global transcriptional effects of elevated c-di-GMP levels, we engineered an RSCV strain that had elevated c-di-GMP levels but did not autoaggregate. Our results showed that about 50 genes are differentially expressed in response to elevated intracellular c-di-GMP levels. Among these genes are the pel and psl genes, which are upregulated, and flagellum and pilus genes, which are downregulated. RSCV traits such as increased exopolysaccharide production leading to antibiotic tolerance, altered metabolism, and reduced immunogenicity may contribute to increased persistence in biofilms and in the airways of CF lungs.

Characterization of Colony Morphology Variants Isolated from Pseudomonas aeruginosa Biofilms

ABSTRACT In this study, we report the isolation of small, rough, strongly cohesive colony morphology variants from aging Pseudomonas aeruginosa PAO1 biofilms. Similar to many of the P. aeruginosa colony morphology variants previously described in the literature, these variants autoaggregate in liquid culture and hyperadhere to solid surfaces. They also exhibit increased hydrophobicity and reduced motility compared to the wild-type parent strain. Despite the similarities in appearance of our colony morphology variant isolates on solid medium, the isolates showed a range of responses in various phenotypic assays. These variants form biofilms with significant three-dimensional structure and more biomass than the wild-type parent. To further explore the nature of the variants, their transcriptional profiles were evaluated. The variants generally showed increased expression of the psl and pel loci, which have been previously implicated in the adherence of P. aeruginosa to solid surfaces. When a mutation in the psl locus was introduced into a colony morphology variant, the colony morphology was only partially affected, but hyperadherence and autoaggregation were lost. Finally, similar colony morphology variants were found in isolates from cystic fibrosis patients. These variants displayed many of the same characteristics as the laboratory variants, suggesting a link between laboratory and cystic fibrosis biofilms.

Does Pseudomonas aeruginosa use intercellular signalling to build biofilm communities

Pseudomonas aeruginosa is a Gram‐negative bacterial species that causes several opportunistic human infections. This organism is also found in the environment, where it is renowned (like other Pseudomonads) for its ability to use a wide variety of compounds as carbon and energy sources. It is a model species for studying group‐related behaviour in bacteria. Two types of group behaviour it engages in are intercellular signalling, or quorum sensing, and the formation of surface‐associated communities called biofilms. Both quorum sensing and biofilm formation are important in the pathogenesis of P. aeruginosa infections. Quorum sensing regulates the expression of several secreted virulence factors and quorum sensing mutant strains are attenuated for virulence in animal models. Biofilms have been implicated in chronic infections. Two examples are the chronic lung infections afflicting people suffering from cystic fibrosis and colonization of indwelling medical devices. This review will discuss quorum sensing and biofilm formation and studies that link these two processes.

The effect of roofing material on the quality of harvested rainwater

Due to decreases in the availability and quality of traditional water resources, harvested rainwater is increasingly used for potable and non-potable purposes. In this study, we examined the effect of conventional roofing materials (i.e., asphalt fiberglass shingle, Galvalume(®) metal, and concrete tile) and alternative roofing materials (i.e., cool and green) on the quality of harvested rainwater. Results from pilot-scale and full-scale roofs demonstrated that rainwater harvested from any of these roofing materials would require treatment if the consumer wanted to meet United States Environmental Protection Agency primary and secondary drinking water standards or non-potable water reuse guidelines; at a minimum, first-flush diversion, filtration, and disinfection are recommended. Metal roofs are commonly recommended for rainwater harvesting applications, and this study showed that rainwater harvested from metal roofs tends to have lower concentrations of fecal indicator bacteria as compared to other roofing materials. However, concrete tile and cool roofs produced harvested rainwater quality similar to that from the metal roofs, indicating that these roofing materials also are suitable for rainwater harvesting applications. Although the shingle and green roofs produced water quality comparable in many respects to that from the other roofing materials, their dissolved organic carbon concentrations were very high (approximately one order of magnitude higher than what is typical for a finished drinking water in the United States), which might lead to high concentrations of disinfection byproducts after chlorination. Furthermore the concentrations of some metals (e.g., arsenic) in rainwater harvested from the green roof suggest that the quality of commercial growing media should be carefully examined if the harvested rainwater is being considered for domestic use. Hence, roofing material is an important consideration when designing a rainwater catchment.

The reduction of bromate by granular activated carbon

Abstract The removal of bromate, an inorganic disinfection by-product, by granular activated carbon (GAC) was investigated in this study. Bromate ion removal from water was observed in the presence of virgin and acid-washed outgassed (AWOG) GAC. In a GAC filter with distilled-deionized water, bromate breakthrough occurred slowly whereas bromate breakthrough occurred very quickly in natural water due to the presence of natural organic matter (NOM) and other anions. NOM adsorption decreased bromate reduction, presumably by blocking bromate reduction sites. The use of a biologically active carbon (BAC) filter with ozonated water, as a pretreatment step to remove NOM, only slightly improved bromate reduction in the subsequent fresh GAC filter. Kinetic studies showed that the presence of chloride, sulfate, bromide, and nitrate causes a decrease in the kinetics of bromate reduction by GAC. These anions may occupy ion exchange sites on the carbon, reducing the rate at which bromate can access the reduction sites. However, when the anions were released from the carbon, the bromate reduction rate increased.

Effects of Chloride and Ionic Strength on Physical Morphology, Dissolution, and Bacterial Toxicity of Silver Nanoparticles

In this study, we comprehensively evaluate chloride- and ionic-strength-mediated changes in the physical morphology, dissolution, and bacterial toxicity of silver nanoparticles (AgNPs), which are one of the most-used nanomaterials. The findings isolate the impact of ionic strength from that of chloride concentration. As ionic strength increases, AgNP aggregation likewise increases (such that the hydrodynamic radius [HR] increases), fractal dimension (Df) strongly decreases (providing increased available surface relative to suspensions with higher Df), and the release of Ag(aq) increases. With increased Ag(+) in solution, Escherichia coli demonstrates reduced tolerance to AgNP exposure (i.e., toxicity increases) under higher ionic strength conditions. As chloride concentration increases, aggregates are formed (HR increases) but are dominated by AgCl(0)(s) bridging of AgNPs; relatedly, Df increases. Furthermore, AgNP dissolution strongly increases under increased chloride conditions, but the dominant, theoretical, equilibrium aqueous silver species shift to negatively charged AgClx((x-1)-) species, which appear to be less toxic to E. coli. Thus, E. coli demonstrates increased tolerance to AgNP exposure under higher chloride conditions (i.e., toxicity decreases). Expression measurements of katE, a gene involved in catalase production to alleviate oxidative stress, support oxidative stress in E. coli as a result of Ag(+) exposure. Overall, our work indicates that the environmental impacts of AgNPs must be evaluated under relevant water chemistry conditions.

WATER QUALITY FACTORS AFFECTING BROMATE REDUCTION IN BIOLOGICALLY ACTIVE CARBON FILTERS

Biological removal of the ozonation by-product, bromate, was demonstrated in biologically active carbon (BAC) filters. For example, with a 20-min EBCT, pH 7.5, and influent dissolved oxygen (DO) and nitrate concentrations 2.1 and 5.1 mg/l, respectively, 40% bromate removal was obtained with a 20 microg/l influent bromate concentration. In this study, DO, nitrate and sulfate concentrations, pH, and type of source water were evaluated for their effect on bromate removal in a BAC filter. Bromate removal decreased as the influent concentrations of DO and nitrate increased, but bromate removal was observed in the presence of measurable effluent concentrations of DO and nitrate. In contrast, bromate removal was not sensitive to the influent sulfate concentration, with only a slight reduction in bromate removal as the influent sulfate concentration was increased from 11.1 to 102.7 mg/l. Bromate reduction was better at lower pH values (6.8 and 7.2) than at higher pH values (7.5 and 8.2), suggesting that it may be possible to reduce bromate formation during ozonation and increase biological bromate reduction through pH control. Biological bromate removal in Lake Michigan water was very poor as compared to that in tapwater from a groundwater source. Bromate removal improved when sufficient organic electron donor was added to remove the nitrate and DO present in the Lake Michigan water, indicating that the poor biodegradability of the natural organic matter may have been limiting bromate removal in that water. Biological bromate removal was demonstrated to be a sustainable process under a variety of water quality conditions, and bromate removal can be improved by controlling key water quality parameters.

Influence of the Hydrodynamic Environment on Quorum Sensing in Pseudomonas aeruginosa Biofilms

We provide experimental and modeling evidence that the hydrodynamic environment can impact quorum sensing (QS) in a Pseudomonas aeruginosa biofilm. The amount of biofilm biomass required for full QS induction of the population increased as the flow rate increased.

Characterization of bromate-reducing bacterial isolates and their potential for drinking water treatment.

The objective of the current study was to isolate and characterize several bromate-reducing bacteria and to examine their potential for bioaugmentation to a drinking water treatment process. Fifteen bromate-reducing bacteria were isolated from three sources. According to 16S rRNA gene sequencing, the bromate-reducing bacteria are phylogenetically diverse, representing the Actinobacteria, Bacteroidetes, Firmicutes, and α-, β-, and γ-Proteobacteria. The broad diversity of bromate-reducing bacteria suggests the widespread capability for microbial bromate reduction. While the cometabolism of bromate via nitrate reductase and (per)chlorate reductase has been postulated, five of our bromate-reducing isolates were unable to reduce nitrate or perchlorate. This suggests that a bromate-specific reduction pathway might exist in some microorganisms. Bioaugmentation of activated carbon filters with eight of the bromate-reducing isolates did not significantly decrease start-up time or increase bromate removal as compared to control filters. To optimize bromate reduction in a biological drinking water treatment process, the predominant mechanism of bromate reduction (i.e., cometabolic or respiratory) needs to be assessed so that appropriate measures can be taken to improve bromate removal.

Prokaryotic suppression subtractive hybridization PCR cDNA subtraction, a targeted method to identify differentially expressed genes.

ABSTRACT Molecular biology tools can be used to monitor and optimize biological treatment systems, but the application of nucleic acid-based tools has been hindered by the lack of available sequences for environmentally relevant biodegradation genes. The objective of our work was to extend an existing molecular method for eukaryotes to prokaryotes, allowing us to rapidly identify differentially expressed genes for subsequent sequencing. Suppression subtractive hybridization (SSH) PCR cDNA subtraction is a technique that can be used to identify genes that are expressed under specific conditions (e.g., growth on a given pollutant). While excellent methods for eukaryotic SSH PCR cDNA subtraction are available, to our knowledge, no methods previously existed for prokaryotes. This work describes our methodology for prokaryotic SSH PCR cDNA subtraction, which we validated using a model system: Pseudomonas putida mt-2 degrading toluene. cDNA from P. putida mt-2 grown on toluene (model pollutant) or acetate (control substrate) was subjected to our prokaryotic SSH PCR cDNA subtraction protocol to generate subtraction clone libraries. Over 90% of the sequenced clones contained gene fragments encoding toluene-related enzymes, and 20 distinct toluene-related genes from three key operons were sequenced. Based on these results, prokaryotic SSH PCR cDNA subtraction shows promise as a targeted method for gene identification.