Proteomic Analyses of Chlorhexidine Tolerance Mechanisms in Delftia acidovorans Biofilms.

Protein expression and fatty acid profiles of biofilm cells of chlorhexidine-tolerant Delftia acidovorans (MIC = 15 µg/ml) and its chlorhexidine-susceptible mutant (MIC = 1 µg/ml) were investigated. The chlorhexidine-susceptible mutant (MT51) was derived from the parental strain (WT15) using Tn5 transposon mutagenesis. The disrupted gene was identified as tolQ, a component of the tolQRAB gene cluster known to be involved in outer membrane stability. Proteomic responses of biofilm cells were compared by differential in-gel electrophoresis following exposure to chlorhexidine at sub-MIC (10 µg/ml) and above-MIC (30 µg/ml) concentrations. Numerous changes in protein abundance were observed in biofilm cells following chlorhexidine exposure, suggesting that molecular changes occurred during adaptation to chlorhexidine. Forty proteins showing significant differences (≥1.5-fold; P < 0.05) were identified by mass spectrometry and were associated with various functions, including amino acid and lipid biosynthesis, protein translation, energy metabolism, and stress-related functions (e.g., GroEL, aspartyl/glutamyl-tRNA amidotransferase, elongation factor Tu, Clp protease, and hydroxymyristoyl-ACP dehydratase). Several proteins involved in fatty acid synthesis were affected by chlorhexidine, in agreement with fatty acid analysis, wherein chlorhexidine-induced shifts in the fatty acid profile were observed in the chlorhexidine-tolerant cells, primarily the cyclic fatty acids. Transmission electron microscopy revealed more prominent changes in the cell envelope of chlorhexidine-susceptible MT51 cells. This study suggests that multiple mechanisms involving both the cell envelope (and likely TolQ) and panmetabolic regulation play roles in chlorhexidine tolerance in D. acidovorans. IMPORTANCE Delftia acidovorans has been associated with a number of serious infections, including bacteremia, empyema, bacterial endocarditis, and ocular and urinary tract infections. It has also been linked with a variety of surface-associated nosocomial infections. Biofilm-forming antimicrobial-resistant D. acidovorans strains have also been isolated, including ones displaying resistance to the common broad-spectrum agent chlorhexidine. The mechanisms of chlorhexidine resistance in D. acidovorans are not known; hence, a chlorhexidine-susceptible mutant of the tolerant wild-type strain was obtained using transposon mutagenesis, and the proteome and ultrastructural changes of both strains were compared under chlorhexidine challenge.

Characterizing manufactured nanoparticles in the environment: multimethod determination of particle sizes.

Sizes of stabilized (24 h) nanoparticle suspensions were determined using several state-of-the-art analytical techniques (transmission electron microscopy; atomic force microscopy; dynamic light scattering; fluorescence correlation spectroscopy; nanoparticle tracking analysis; flow field flow fractionation). Theoretical and analytical considerations were evaluated, results were compared, and the advantages and limitations of the techniques were discussed. No "ideal" technique was found for characterizing manufactured nanoparticles in an environmental context as each technique had its own advantages and limitations.

Morphological and biochemical changes in Pseudomonas fluorescens biofilms induced by sub-inhibitory exposure to antimicrobial agents.

Confocal laser scanning microscopy (CLSM) and scanning transmission X-ray microscopy (STXM) were used to examine the morphological and biochemical changes in Pseudomonas fluorescens biofilms grown in the presence of subinhibitory concentrations of 4 antimicrobial agents: triclosan, benzalkonium chloride, chlorhexidine dihydrochloride, and trisodium phosphate. CLSM analyses using the stains SYTO9 and propidium iodide indicated that the antimicrobial agents affected cell membrane integrity and cellular density to differing degrees. However, fluorescein diacetate assays and plate counts demonstrated that the cells remained metabolically active. Fluorescent lectin binding assays showed that changes in the arrangement and composition of the exopolymer matrix of the biofilms also occurred and that these changes depended on the antimicrobial agent. Detailed single cell analyses using STXM provided evidence that the cell morphology, and the spatial distribution and relative amounts of protein, lipids and polysaccharides in the biofilms and within the cells were different for each antimicrobial. The distribution of chlorhexidine in the biofilm, determined from its distinct spectral signature, was localized mainly inside the bacterial cells. Each antimicrobial agent elicited a unique response; P. fluorescens cells and biofilms changed their morphology and architecture, as well as the distribution and abundance of biomacromolecules, in particular the exopolymer matrix. Pseudomonas fluorescens also exhibited adaptation to benzalkonium chloride at 10 microg/mL. Our observations point to the importance of changes in the quantity and chemistry of the exopolymeric matrix in the response to antimicrobial agents and suggest their importance as targets for control.

Quantitative mapping of chlorhexidine in natural river biofilms.

Soft X-ray scanning transmission X-ray microscopy has been applied to map chlorhexidine, a ubiquitous antimicrobial agent, relative to major biochemical components (proteins, lipids, polysaccharides, Ca2+, K+, CO3(2-)) in natural river biofilms. For the first time, bio-accumulation of chlorhexidine in diatoms has been observed unambiguously. The quantitative results show that chlorhexidine bioaccumulated extensively in lipid-rich regions of diatoms and bacteria. Confocal laser scanning microscopy was used to document changes in the biofilm community. The bioaccumulation provides a significant entry point for chlorhexidine into the aquatic food chain. It results in modification of the biofilm community and it impacts the photosynthetic and protozoan species in particular. X-ray microscopy mapping at high spatial resolution is shown to be a powerful tool for studies of antimicrobial agents in the environment.

Speciation and quantitative mapping of metal species in microbial biofilms using scanning transmission X-ray microscopy.

A scanning transmission X-ray microscope illuminated with synchrotron light was used to investigate the speciation and spatial distributions of metals in a microbial biofilm cultivated from river water. Metal 2p absorption edge signals were used to provide metal speciation (through shapes of the absorption spectra) and quantitative spatial distributions of the metal species. This paper presents sample data and describes methods for extracting quantitative maps of metal species from image sequences recorded in the region of the metal 2p edges. Comparisons were made with biochemical characterization of the same region using images recorded at the C 1s and O 1s edges. The method is applied to detailed quantitative analysis of ferrous and ferric iron in a river biofilm, in concert with mapping Ni(II) and Mn(II) species in the same region. The distributions of the metal species are discussed in the context of the biofilm structure. These results demonstrate that soft X-ray STXM measurements at the metal 2p absorption edges can be used to speciate metals and to provide quantitative spatial distribution maps for metal species in environmental samples with 50 nm spatial resolution.

Refined tunable methodology for characterization of contaminant-particle relationships in surface water.

To understand contaminant transport in aquatic systems, it is essential to define the physical characteristics of the primary particulate carriers. The distribution of organic pollutants with particle-size class and particle morphology in a freshwater embayment (Hamilton Harbor, western Lake Ontario) was studied using a sequence of novel sample preparation and characterization techniques. Water samples (24 L) were fractionated according to particle-size distribution using differential cascade sedimentation and centrifugation methods. These size fractions were subsequently subjected to a physicochemical characterization using scanning transmission electron microscopy and energy-dispersive spectroscopy to identify flocs and individual colloidal particles in the size range of 1 nm to 1 mm in diameter. Analytical chemical analyses were performed to identify organic contaminants in extracts prepared from particle-size classes, including polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs). The contaminant distribution trends were very similar for all compound classes studied; contaminants were primarily associated with fractions containing particles less than 2 mum in diameter. Morphological characterization of these fractions showed the majority of the particulates to be humic fractals. The results of this study show that contaminants in aquatic systems can be preferentially associated with specific types of particle carriers, the characteristics of which can be clearly defined in terms of size and morphology.

Compartmentalization of metals within the diverse colloidal matrices comprising activated sludge microbial flocs.

Activated sludge floc from a wastewater treatment system was characterized, with regard to principal structural, chemical, and microbiological components and properties, in relation to contaminant-colloid associations and settling. Multiscale analytical microscopies, in conjunction with multimethod sample preparations, were used correlatively to characterize diverse colloidal matrices within microbial floc. Transmission electron microscopy, in conjunction with energy dispersive spectroscopy (EDS), revealed specific associations of contaminant heavy metals with individual bacterial cells and with extracellular polymeric substances (EPS). Floc structure was mapped from the level of gross morphology down to the nano-scale, and flocs were described with respect to settling properties, size, shape, density, porosity, bound water content, and EPS chemical composition; gross surface properties were also measured for correlation with principal floc features. Compartmentalization results based on 171 EDS analyses and representative high-resolution images showed that nano-scale agglomerations of (i) silver (100%) and (ii) zinc (91%) were confined almost entirely to EPS matrices while (iii) Pb (100%) was confined to intracellular granules and (iv) aluminum was partitioned between EPS matrices (41%) and intracellular matrices (59%). The results suggest that engineered changes in microbial physiology and/or in macromolecular EPS composition may influence metal removal efficiencies.