New Device for High-Throughput Viability Screening of Flow Biofilms

Control of biofilms requires rapid methods to identify compounds effective against them and to isolate resistance-compromised mutants for identifying genes involved in enhanced biofilm resistance. While rapid screening methods for microtiter plate well (“static”) biofilms are available, there are no methods for such screening of continuous flow biofilms (“flow biofilms”). Since the latter biofilms more closely approximate natural biofilms, development of a high-throughput (HTP) method for screening them is desirable. We describe here a new method using a device comprised of microfluidic channels and a distributed pneumatic pump (BioFlux) that provides fluid flow to 96 individual biofilms. This device allows fine control of continuous or intermittent fluid flow over a broad range of flow rates, and the use of a standard well plate format provides compatibility with plate readers. We show that use of green fluorescent protein (GFP)-expressing bacteria, staining with propidium iodide, and measurement of fluorescence with a plate reader permit rapid and accurate determination of biofilm viability. The biofilm viability measured with the plate reader agreed with that determined using plate counts, as well as with the results of fluorescence microscope image analysis. Using BioFlux and the plate reader, we were able to rapidly screen the effects of several antimicrobials on the viability of Pseudomonas aeruginosa PAO1 flow biofilms.Bacterial biofilms are surface-attached communities that are encased in a polymeric matrix, which exhibit a high degree of resistance to antimicrobial agents and the host immune system (12, 16). This makes them medically important; diseases with a biofilm component are chronic and difficult to eradicate. Examples of such diseases are cystitis (1), endocarditis (31), cystic fibrosis (35), and middle-ear (17) and indwelling medical device-associated (20) infections. Biofilms also play important environmental roles in, for example, wastewater treatment (38), bioremediation (29, 30), biofouling (7), and biocorrosion (2). Better control of biofilms requires elucidation of the molecular basis of their superior resistance (by identifying resistance-compromised mutants) and identification of compounds with antibiofilm activity. While our understanding of these aspects of biofilms has increased (11, 15, 25-27, 36), further work, including development of accurate high-throughput (HTP) methods for screening biofilm viability, is needed.Two major biofilm models are studied in the laboratory, biofilms grown without a continuous flow of fresh medium and biofilms grown with a continuous flow of fresh medium; examples of these two models are microtiter well biofilms and flow cell biofilms, respectively. Methods have been developed for HTP screening of the viability of static biofilms (6, 28, 32, 33), but there are no methods for HTP screening of flow biofilms. The latter biofilms are typically grown in flow cells, which have to be examined individually to determine viability and thus cannot be used for rapid screening. An HTP screening method for flow biofilms is desirable, as these biofilms more closely approximate natural biofilms and can differ from static biofilms evidently due to hydrodynamic influences on cell signaling (22, 34). For example, the ability of rpoS-deficient Escherichia coli (lacking σ^S) to form flow biofilms is impaired, but its capacity to form biofilms under static conditions is enhanced (18).We describe here a new application of a recently developed device (8-10, 13), the “BioFlux” device consisting of microfluidic channels for biofilm growth. Other microfluidic devices have recently been used for biofilm formation (14, 19, 21, 23), but none of them has been used for HTP screening. The BioFlux device permits rapid measurement of the fluorescence of flow biofilms with a plate reader, which permits initial HTP screening of the viability of such biofilms.     

Immobilization of Cr(VI) and Its Reduction to Cr(III) Phosphate by Granular Biofilms Comprising a Mixture of Microbes

We assessed the potential of mixed microbial consortia, in the form of granular biofilms, to reduce chromate and remove it from synthetic minimal medium. In batch experiments, acetate-fed granular biofilms incubated aerobically reduced 0.2 mM Cr(VI) from a minimal medium at 0.15 mM day⁻¹ g⁻¹, with reduction of 0.17 mM day⁻¹ g⁻¹ under anaerobic conditions. There was negligible removal of Cr(VI) (i) without granular biofilms, (ii) with lyophilized granular biofilms, and (iii) with granules in the absence of an electron donor. Analyses by X-ray absorption near edge spectroscopy (XANES) of the granular biofilms revealed the conversion of soluble Cr(VI) to Cr(III). Extended X-ray absorption fine-structure (EXAFS) analysis of the Cr-laden granular biofilms demonstrated similarity to Cr(III) phosphate, indicating that Cr(III) was immobilized with phosphate on the biomass subsequent to microbial reduction. The sustained reduction of Cr(VI) by granular biofilms was confirmed in fed-batch experiments. Our study demonstrates the promise of granular-biofilm-based systems in treating Cr(VI)-containing effluents and wastewater.Chromium is a common industrial chemical used in tanning leather, plating chrome, and manufacturing steel. The two stable environmental forms are hexavalent chromium [Cr(VI)] and trivalent chromium [Cr(III)] (20). The former is highly soluble and toxic to microorganisms, plants, and animals, entailing mutagenic and carcinogenic effects (6, 22, 33), while the latter is considered to be less soluble and less toxic. Therefore, the reduction of Cr(VI) to Cr(III) constitutes a potential detoxification process that might be achieved chemically or biologically. Microbial reduction of Cr(VI) seemingly is ubiquitous; Cr(VI)-reducing bacteria have been isolated from both Cr(VI)-contaminated and -uncontaminated environments (6, 7, 23, 38, 39). Many archaeal/eubacterial genera, common to different environments, reduce a wide range of metals, including Cr(VI) (6, 16, 21). Some bacterial enzymes generate Cr(V) by mediating one-electron transfer to Cr(VI) (1, 4), while many other chromate reductases convert Cr(VI) to Cr(III) in a single step.Biological treatment of Cr(VI)-contaminated wastewater may be difficult because the metal''s toxicity potentially can kill the bacteria. Accordingly, to protect the cells, cell immobilization techniques were employed (31). Cells in a biofilm exhibit enhanced resistance and tolerance to toxic metals compared with free-living ones (15). Therefore, biofilm-based reduction of Cr(VI) and its subsequent immobilization might be a satisfactory method of bioremediation because (i) the biofilm-bound cells can tolerate higher concentrations of Cr(VI) than planktonic cells, and (ii) they allow easy separation of the treated liquid from the biomass. Ferris et al. (11) described microbial biofilms as natural metal-immobilizing matrices in aqueous environments. Bioflocs, the active biomass of activated sludge-process systems are transformed into dense granular biofilms in sequencing batch reactors (SBRs). As granular biofilms settle extremely well, the treated effluent is separated quickly from the granular biomass by sedimentation (9, 24). Previous work demonstrated that aerobic granular biofilms possess tremendous ability for biosorption, removing zinc, copper, nickel, cadmium, and uranium (19, 26, 31, 32, 40). However, no study has investigated the role of cellular metabolism of aerobically grown granular biofilms in metal removal experiments. Despite vast knowledge about biotransformation by pure cultures, very little is known about reduction and immobilization by mixed bacterial consortia (8, 12, 13, 16, 20, 31, 36). Our research explored, for the first time, the metabolically driven removal of Cr(VI) by microbial granules.The main aim of this study was to investigate Cr(VI) reduction and immobilization by mixed bacterial consortia, viz., aerobically grown granular biofilms. Such biofilm-based systems are promising for developing compact bioreactors for the rapid biodegradation of environmental contaminants (17, 24, 29). Accordingly, we investigated the microbial reduction of Cr(VI) by aerobically grown biofilms in batch and fed-batch experiments and analyzed the oxidation state and association of the chromium immobilized on the biofilms by X-ray absorption near edge spectroscopy (XANES) and extended X-ray absorption fine structure (EXAFS).     

Antibody-Mediated Immobilization of Cryptococcus neoformans Promotes Biofilm Formation

Most microbes, including the fungal pathogen Cryptococcus neoformans, can grow as biofilms. Biofilms confer upon microbes a range of characteristics, including an ability to colonize materials such as shunts and catheters and increased resistance to antibiotics. Here, we provide evidence that coating surfaces with a monoclonal antibody to glucuronoxylomannan, the major component of the fungal capsular polysaccharide, immobilizes cryptococcal cells to a surface support and, subsequently, promotes biofilm formation. We used time-lapse microscopy to visualize the growth of cryptococcal biofilms, generating the first movies of fungal biofilm growth. We show that when fungal cells are immobilized using surface-attached specific antibody to the capsule, the initial stages of biofilm formation are significantly faster than those on surfaces with no antibody coating or surfaces coated with unspecific monoclonal antibody. Time-lapse microscopy revealed that biofilm growth was a dynamic process in which cells shuffled position during budding and was accompanied by emergence of planktonic variant cells that left the attached biofilm community. The planktonic variant cells exhibited mobility, presumably by Brownian motion. Our results indicate that microbial immobilization by antibody capture hastens biofilm formation and suggest that antibody coating of medical devices with immunoglobulins must exclude binding to common pathogenic microbes and the possibility that this effect could be exploited in industrial microbiology.Cryptococcus neoformans is a fungal pathogen that is ubiquitous in the environment and enters the body via the inhalation of airborne particles. The C. neoformans cell is surrounded by a layer of polysaccharide that consists predominantly of glucuronoxylomannan (GXM), which forms a protective capsule around the microbe. The capsule has been shown to be essential for virulence in murine models of infection (5-7) and, thus, is considered a key virulence factor. C. neoformans is the causative agent of cryptococcosis, a disease that primarily affects individuals with impaired immune systems, and is a significant problem in AIDS patients (21, 31). The most common manifestation of cryptococcosis is meningoencephalitis.Biofilms are communities of microbes that are attached to surfaces and held together by an extracellular matrix, often consisting predominantly of polysaccharides (8, 10). A great deal is known about bacterial biofilms (3, 9, 24, 30), but fungal biofilm formation is much less studied. Candida albicans is known to synthesize biofilms (11, 28, 29), as is C. neoformans. Biofilm-like structures consisting of innumerable cryptococcal cells encased in a polysaccharide matrix have been reported in human cases of cryptococcosis (32). Biofilm formation confers upon the microbe the capacity for drug resistance, and microbial cells in biofilms are less susceptible to host defense mechanisms (2, 4, 9, 12). In this regard, cells within C. neoformans biofilms are significantly less susceptible to caspofungin and amphotericin B than are planktonic cells (19). The cells within the biofilm are also resistant to the actions of fluconazole and voriconazole and various microbial oxidants and peptides (17, 19).Bacterial and fungal biofilms form readily on prosthetic materials, which poses a tremendous risk of chronic infection (10, 13, 15, 27). C. neoformans biofilms can form on a range of surfaces, including glass, polystyrene, and polyvinyl, and material devices, such as catheters (16). C. neoformans can form biofilms on the ventriculoatrial shunts used to decompress intracerebral pressure in patients with cryptococcal meningoencephalitis (32).The polysaccharide capsule of C. neoformans is essential for biofilm formation (18), and biofilm formation involves the shedding and accumulation of large amounts of GXM into the biofilm extracellular matrix (16). Previously, we reported that antibody to GXM in solution could inhibit biofilm formation through a process that presumably involves interference with polysaccharide shedding (18, 20). However, the effect of antibody-mediated immobilization of C. neoformans cells on cryptococcal biofilm formation has not been explored. In this paper, we report that the monoclonal antibody (MAb) 18B7, which is specific for the capsular polysaccharide GXM, can capture and immobilize C. neoformans to surfaces, a process that promotes biofilm formation. Interestingly, we identified planktonic variant C. neoformans cells that appeared to escape from the biofilm, but whose functions are not known. The results provide new insights on biofilm formation.     

Evolution of Diversity in Spatially Structured Escherichia coli Populations

The stochastic Ricker population model was used to investigate the generation and maintenance of genetic diversity in a bacterial population grown in a spatially structured environment. In particular, we showed that Escherichia coli undergoes dramatic genetic diversification when grown as a biofilm. Using a novel biofilm entrapment method, we retrieved 64 clones from each of six different depths of a mature biofilm, and after subculturing for ∼30 generations, we measured their growth kinetics in three different media. We fit a stochastic Ricker population growth model to the recorded growth curves. The growth kinetics of clonal lineages descendant from cells sampled at different biofilm depths varied as a function of both the depth in the biofilm and the growth medium used. We concluded that differences in the growth dynamics of clones were heritable and arose during adaptive evolution under local conditions in a spatially heterogeneous environment. We postulate that under nutrient-limited conditions, selective sweeps would be protracted and would be insufficient to purge less-fit variants, a phenomenon that would allow the coexistence of genetically distinct clones. These findings contribute to the current understanding of biofilm ecology and complement current hypotheses for the maintenance and generation of microbial diversity in spatially structured environments.The mechanisms that lead to the genesis and maintenance of diversity in communities have intrigued geneticists and ecologists alike for decades (6, 17, 27, 33, 39, 49). This is particularly challenging for microbial communities, in which ecological and evolutionary processes occur on roughly the same time scale (3, 16, 38) and where the outcome of these processes may be affected by the spatial structure in which these communities grow.Bacterial biofilms are examples of spatially structured communities that have been the subject of intense research in medical and engineering contexts in recent years (3, 8, 20, 48, 56). Previous work has shown that the phenotypic characteristics of bacterial populations in biofilms are distinct from those of their free-swimming counterparts (8). These bacterial assemblages form physically and chemically heterogeneous structures (20) whose complex architecture strongly influences mass transfer (56). This results in the formation of steep gradients of nutrients, waste products, pH, redox potential, and electron acceptors, which results in the creation of distinct and perhaps unique niches on a microscale. This places selective pressure on variants that have enhanced fitness and are well adapted to local conditions. From a theoretical perspective, this would be expected to increase genetic diversity within a population by precluding competitive exclusion, yet this has not previously been demonstrated empirically.The degree of diversification that occurs within populations growing in biofilms is not well understood, nor are the spatial and temporal dynamics of bacterial species succession in biofilms. However, it is known that the physical and chemical heterogeneity of microbial biofilms has profound effects on microbial growth and activity. Most bacterial cells in biofilms are not highly active and grow slowly if at all. For example, active protein synthesis occurs only in the uppermost zone (32 ± 3 μm) of Pseudomonas aeruginosa biofilms (4). Likewise, in Klebsiella pneumoniae biofilms, fast growth occurs near the interface of the biofilm and bulk fluid, and cells inside the biofilm show little growth (55). The near absence of growth in interior regions of biofilms may lead to an increased tempo of diversification, since numerous studies have shown that mutation frequencies are elevated in slowly growing cells (28). If this occurs within a biofilm, then clones might exhibit a high genotypic variability that could have significant practical implications in terms of yielding spontaneous mutants that are resistant to antimicrobial agents.Experimental evolution has contributed greatly to our understanding of the causes and consequences of genetic diversity in populations (reviewed in references 23, 29, and 42). Initially, research focused on characterizing diversity within populations that evolved in spatially homogenous environments (e.g., chemostat and batch systems) (13, 15, 19, 30-32, 45, 47, 50-53). Several studies have highlighted a role for spatial heterogeneity in the emergence and maintenance of genetic diversity (25, 26, 43). Korona and colleagues (25, 26) compared populations that evolved in batch cultures to populations that evolved with a spatial structure and demonstrated that phenotypic diversity was greatest with spatial structure. In other work, Rainey and Travisano (43) showed that populations of Pseudomonas grown in static broth microcosms diversified so that some ecotypes occupied a floating biofilm on the surface of the broth while others occupied the liquid phase or glass surface of the culture. Boles et al. (2, 3) investigated the extent of diversification of Pseudomonas using biofilms that evolved in flow-cell systems. They reported that genetic changes produced by a recA-dependent mechanism affected multiple traits, with some biofilm-derived variants being better able to disseminate while others were better able to form biofilms (3). Further study showed that in some cells, endogenous oxidative stress caused double-stranded DNA breaks that when repaired by recombinatorial DNA repair genes gave rise to mutations (2). These previous studies demonstrate the pivotal role of spatial structure in the generation and maintenance of diversity in evolving bacterial populations.In this study, we extended this work by using novel techniques to characterize diversity in Escherichia coli biofilms that allowed us to recover clones from specific depths within a biofilm. The growth kinetics of clones from six different biofilm depths were measured and modeled using an analysis-of-variance formulation of the stochastic Ricker model of population dynamics with environmental noise (11, 40). Rigorous statistical methods were used to show that after 1 month of cultivation, the extant diversity in E. coli biofilms was extraordinarily high and varied with depth.     

Increased Antibiotic Resistance of Escherichia coli in Mature Biofilms

Biofilms are considered to be highly resistant to antimicrobial agents. Several mechanisms have been proposed to explain this high resistance of biofilms, including restricted penetration of antimicrobial agents into biofilms, slow growth owing to nutrient limitation, expression of genes involved in the general stress response, and emergence of a biofilm-specific phenotype. However, since combinations of these factors are involved in most biofilm studies, it is still difficult to fully understand the mechanisms of biofilm resistance to antibiotics. In this study, the antibiotic susceptibility of Escherichia coli cells in biofilms was investigated with exclusion of the effects of the restricted penetration of antimicrobial agents into biofilms and the slow growth owing to nutrient limitation. Three different antibiotics, ampicillin (100 μg/ml), kanamycin (25 μg/ml), and ofloxacin (10 μg/ml), were applied directly to cells in the deeper layers of mature biofilms that developed in flow cells after removal of the surface layers of the biofilms. The results of the antibiotic treatment analyses revealed that ofloxacin and kanamycin were effective against biofilm cells, whereas ampicillin did not kill the cells, resulting in regrowth of the biofilm after the ampicillin treatment was discontinued. LIVE/DEAD staining revealed that a small fraction of resistant cells emerged in the deeper layers of the mature biofilms and that these cells were still alive even after 24 h of ampicillin treatment. Furthermore, to determine which genes in the biofilm cells are induced, allowing increased resistance to ampicillin, global gene expression was analyzed at different stages of biofilm formation, the attachment, colony formation, and maturation stages. The results showed that significant changes in gene expression occurred during biofilm formation, which were partly induced by rpoS expression. Based on the experimental data, it is likely that the observed resistance of biofilms can be attributed to formation of ampicillin-resistant subpopulations in the deeper layers of mature biofilms but not in young colony biofilms and that the production and resistance of the subpopulations were aided by biofilm-specific phenotypes, like slow growth and induction of rpoS-mediated stress responses.Reduced susceptibility of biofilm bacteria to antimicrobial agents is a crucial problem for treatment of chronic infections (11, 29, 48). It has been estimated that 65% of microbial infections are associated with biofilms (11, 29, 37), and biofilm cells are 100 to 1,000 times more resistant to antimicrobial agents than planktonic bacterial cells (11, 29, 32).The molecular nature of this apparent resistance has not been elucidated well, and a number of mechanisms have been proposed to explain the reduced susceptibility, such as restricted antibiotic penetration (47), decreased growth rates and metabolism (7, 52), quorum sensing and induction of a biofilm-specific phenotype (8, 29, 35, 39, 49), stress response activation (7, 52), and an increase in expression of efflux pumps (14). Biofilm resistance has generally been assumed to be due to the fact that the cells in the deeper layers of thick biofilms, which grow more slowly, have less access to antibiotics and nutrients. However, this is not the only reason in many cases. Familiar mechanisms of antibiotic resistance, such as modifying enzymes and target mutations, do not seem to be responsible for the biofilm resistance. Even sensitive bacteria that do not have a known genetic basis for resistance can exhibit profoundly reduced susceptibility when they form biofilms (48).It was reported previously that changes in gene expression induced a biofilm-specific phenotype (5, 13, 22, 35, 41, 42). Several genes have been proposed to be particularly important for biofilm formation, and the importance of the rpoS gene in Escherichia coli biofilm formation was suggested recently (1, 10, 22, 42). It has been suggested that induction of an rpoS-mediated stress response results in physiological changes that could contribute to antibiotic resistance (29). Although several mechanisms and genes have been proposed to explain biofilm resistance to antibiotics, this resistance is not still fully understood because these mechanisms seem to work together within a biofilm community. In addition, the physiology of biofilm cells is remarkably heterogeneous and varies according to the location of individual cells within biofilms (33, 34, 46).In this study, susceptibility of E. coli cells in biofilms to antibiotics was investigated. The E. coli cells in the deeper layers of mature biofilms were directly treated with three antibiotics with different molecular targets, the β-lactam ampicillin, the aminoglycoside kanamycin, and the fluoroquinolone ofloxacin. The biofilm biomass was removed before antibiotic treatment, and only the cells located in the deeper layers of the mature biofilms were directly exposed to antibiotics; thus, the effects of restricted antibiotic and nutrient penetration, as well as heterogeneous physiological states in biofilms, were reduced. Although ofloxacin and kanamycin effectively killed the biofilm cells, ampicillin could not kill the cells, which led to regrowth of biofilms. However, the cells in young colony biofilms were completely killed by ampicillin. Therefore, to determine which genes are induced in the mature biofilm cells, allowing increased resistance to ampicillin, global gene expression was analyzed at different stages of biofilm formation, the attachment, colony formation, and maturation stages. Based on the experimental data obtained, possible mechanisms of the increased biofilm resistance to ampicillin are discussed below.     

Identification of Diverse Antimicrobial Resistance Determinants Carried on Bacterial,Plasmid, or Viral Metagenomes from an Activated Sludge Microbial Assemblage

Using both sequence- and function-based metagenomic approaches, multiple antibiotic resistance determinants were identified within metagenomic libraries constructed from DNA extracted from bacterial chromosomes, plasmids, or viruses within an activated sludge microbial assemblage. Metagenomic clones and a plasmid that in Escherichia coli expressed resistance to chloramphenicol, ampicillin, or kanamycin were isolated, with many cloned DNA sequences lacking any significant homology to known antibiotic resistance determinants.Activated sludge in wastewater treatment plants is an open system with a dynamic and phylogenetically diverse microbial community (2, 3, 6, 7, 10, 11). Since the activated sludge process promotes cellular interactions among diverse microorganisms, there is great potential for the lateral transfer of antibiotic resistance genes between microbes in activated sludge and in downstream environments. Several studies have previously identified antibiotic resistance determinants from wastewater communities that are carried on bacterial chromosomes (1, 4, 14) and plasmids (9, 12, 13), but to our knowledge, a simultaneous metagenomic survey of antibiotic resistance determinants from all three genetic reservoirs (i.e., chromosomes, plasmids, and viruses) has never been performed within the same environment. To achieve a more comprehensive assessment of antibiotic resistance genes in the activated sludge microbial community, this study used both function- and sequence-based metagenomic approaches to identify antibiotic resistance determinants carried on bacterial chromosomes, plasmids, or viruses within an activated sludge microbial assemblage.     

Census of the Viral Metagenome within an Activated Sludge Microbial Assemblage

The viral metagenome within an activated sludge microbial assemblage was sampled using culture-dependent and culture-independent methods and compared to the diversity of activated sludge bacterial taxa. A total of 70 unique cultured bacterial isolates, 24 cultured bacteriophages, 829 bacterial metagenomic clones of 16S rRNA genes, and 1,161 viral metagenomic clones were subjected to a phylogenetic analysis.Bacteriophages play an active role in the ecology of natural environments, influencing prokaryotic population dynamics (5, 15) and mediating lateral gene transfer between diverse bacterial species, for example. Activated sludge (AS) microbial assemblages in wastewater treatment plants have been shown to harbor great numbers of viruses with a wide range of genome sizes (7, 9, 10, 16). Historically, the focus of wastewater viral studies has been on specific host-virus interactions, the application of phages as tools in microbial source tracking, or the use of phages to improve the efficiency of the wastewater treatment process (e.g., foam and pathogen control) (2, 4, 8, 12, 17). Despite the interest in the wastewater viral community, a census of the activated sludge total viral community has not, to our knowledge, been investigated using both culture-based and metagenomic approaches.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

Competition of Listeria monocytogenes Serotype 1/2a and 4b Strains in Mixed-Culture Biofilms

The majority of Listeria monocytogenes isolates recovered from foods and the environment are strains of serogroup 1/2, especially serotypes 1/2a and 1/2b. However, serotype 4b strains cause the majority of human listeriosis outbreaks. Our investigation of L. monocytogenes biofilms used a simulated food-processing system that consisted of repeated cycles of growth, sanitation treatment, and starvation to determine the competitive fitness of strains of serotypes 1/2a and 4b in pure and mixed-culture biofilms. Selective enumeration of strains of a certain serotype in mixed-culture biofilms on stainless steel coupons was accomplished by using serotype-specific quantitative PCR and propidium monoazide treatment to prevent amplification of extracellular DNA or DNA from dead cells. The results showed that the serotype 1/2a strains tested were generally more efficient at forming biofilms and predominated in the mixed-culture biofilms. The growth and survival of strains of one serotype were not inhibited by strains of the other serotype in mixed-culture biofilms. However, we found that a cocktail of serotype 4b strains survived and grew significantly better in mixed-culture biofilms containing a specific strain of serotype 1/2a (strain SK1387), with final cell densities averaging 0.5 log₁₀ CFU/cm² higher than without the serotype 1/2a strain. The methodology used in this study contributed to our understanding of how environmental stresses and microbial competition influence the survival and growth of L. monocytogenes in pure and mixed-culture biofilms.A prominent food-borne pathogen, Listeria monocytogenes can cause severe infections in humans, primarily in high-risk populations, though the disease (listeriosis) is relatively rare (11, 30, 43). Outbreaks of listeriosis have resulted from the contamination of a variety of foods by L. monocytogenes, especially meat and dairy products (27). L. monocytogenes is ubiquitous in the environment, able to grow at refrigeration temperature, and tolerant of the low pHs (3 to 4) typical of acidified foods (28, 32, 44). The capacity to produce biofilms confers protection against stresses common in the food-processing environment (13, 33).Biofilms are characterized by dense clusters of bacterial cells embedded in extracellular polymeric substances which are secreted by cells to aid in adhesion to surfaces and to other cells (4, 5). Strains of L. monocytogenes have been known to persist for years in food-processing environments, presumably in biofilms. Of the 13 known serotypes of L. monocytogenes, three (1/2a, 1/2b, and 4b) account for >95% of the isolates from human illness (21). Serotype 1/2a accounts for >50% of the L. monocytogenes isolates recovered from foods and the environment, while most major outbreaks of human listeriosis have been caused by serotype 4b strains (1, 3, 14, 15, 17, 22, 29, 31, 41, 47, 49,). No correlation between L. monocytogenes strain fitness and serotype has been identified (16, 19). Some studies have reported that strains repeatedly isolated from food and environmental samples (defined as persistent strains) had a higher adherence capacity than strains that were sporadically isolated (2, 36), while this phenomenon was not observed by others (7). Serotype 4b strains exhibited a higher capacity for biofilm formation than did serotype 1/2a strains (36), whereas this was not observed by Di Bonaventura and colleagues (6). It has been suggested that serotype 1/2a strains could be more robust than serotype 4b strains in biofilm formation under a variety of environmental conditions. Furthermore, strains of these serotypes differ in terms of the medium that promotes biofilm formation. Biofilm formation by serotype 4b strains was higher in full-strength tryptic soy broth than in diluted medium, whereas the opposite was observed with serotype 1/2a strains, which produced more biofilm in diluted medium (12).There is limited information on microbial competition between strains of different serotypes in biofilms or on how the environmental stresses present in food-processing environments may affect the biofilm formation and survival of L. monocytogenes of different serotypes. In food-processing plants, the environmental stresses encountered by bacteria are more complex and variable than most laboratory systems used for microbial ecology and biofilm studies. A simulated food-processing (SFP) system has been developed to address this issue (38). The SFP system incorporates several stresses that may affect bacteria in biofilms in the food-processing environment, including exposure to sanitizing agents, dehydration, and starvation. When biofilms were subjected to the SFP regimen over a period of several weeks, the cell numbers of L. monocytogenes strains in the biofilms initially were reduced and then increased as the culture adapted (38). The development of resistance to sanitizing agents was specific to the biofilm-associated cells and was not apparent in the detached cells (38). This suggested that extracellular polymeric substances present in the biofilm matrix were responsible for the resistance to sanitizing agents. It was subsequently found that real-time PCR, in combination with propidium monoazide (PMA) treatment of samples prior to DNA isolation, was an effective method for enumerating viable cells in biofilms (37).The objective of this study was to determine if strains of serotype 1/2a or 4b have a selective advantage under stress conditions. We investigated and compared the initial attachment and biofilm formation capabilities of L. monocytogenes strains of these two serotypes and analyzed the survival and growth of bacteria of each serotype in mixed-serotype biofilms in the SFP system by using PMA with quantitative PCR.     

Proteorhodopsin-Bearing Bacteria in Antarctic Sea Ice

Proteorhodopsins (PRs) are widespread bacterial integral membrane proteins that function as light-driven proton pumps. Antarctic sea ice supports a complex community of autotrophic algae, heterotrophic bacteria, viruses, and protists that are an important food source for higher trophic levels in ice-covered regions of the Southern Ocean. Here, we present the first report of PR-bearing bacteria, both dormant and active, in Antarctic sea ice from a series of sites in the Ross Sea using gene-specific primers. Positive PR sequences were generated from genomic DNA at all depths in sea ice, and these sequences aligned with the classes Alphaproteobacteria, Gammaproteobacteria, and Flavobacteria. The sequences showed some similarity to previously reported PR sequences, although most of the sequences were generally distinct. Positive PR sequences were also observed from cDNA reverse transcribed from RNA isolated from sea ice samples. This finding indicates that these sequences were generated from metabolically active cells and suggests that the PR gene is functional within sea ice. Both blue-absorbing and green-absorbing forms of PRs were detected, and only a limited number of blue-absorbing forms were found and were in the midsection of the sea ice profile in this study. Questions still remain regarding the protein''s ecological functions, and ultimately, field experiments will be needed to establish the ecological and functional role of PRs in the sea ice ecosystem.Proteorhodopsins (PRs) are retinal binding bacterial integral membrane proteins that function as light-driven proton pumps (9, 10) and belong to the microbial rhodopsin superfamily of proteins (54). Since the first reported PR sequence from members of SAR86 clade marine (class Gammaproteobacteria) in 2000 (9), many other PR-bearing bacteria have been identified in a range of marine habitats (5, 18, 20, 24, 25, 46, 62). In the recent Global Ocean Sampling (GOS) expedition, almost 4,000 PR sequences from 41 distinct surface marine environments were acquired, demonstrating that these PR genes are extremely abundant in the genomes of ocean bacterioplankton (46). In fact, PR-containing bacteria account for 13% of the community in the Mediterranean Sea and Red Sea and 70% of the community in the Sargasso Sea (18, 46, 49, 60). These light-harvesting bacteria are present in three major marine classes of bacteria: the Alphaproteobacteria, Gammaproteobacteria, and Flavobacteria. In addition, two distinct PR genes encode pigments with “blue-absorbing” and “green-absorbing” properties, which is achieved by a substitution at a single amino acid position, which thereby functions as a spectral tuning switch (10, 37, 48).Sea ice represents a complex physicochemical environment in polar regions and covers up to 13% of the Earth''s surface (59). Although extreme gradients of temperature, salinity, nutrient availability, and light stratify the ice matrix from the surface to the ice-water interface (41), the sea ice habitat nevertheless supports a diverse microbial community of phytoplankton, Bacteria, Archaea, viruses, and protists that grow in liquid brine channels within the ice (14, 35, 56). This sea ice microbial community (SIMCO) is highly metabolically active despite being unable to avoid the extreme environmental conditions that they experience (39). In fact, very-high-standing stocks of the SIMCO exist in many regions of the Southern Ocean. For example, the concentration of chlorophyll a, a proxy for microalgal biomass, typically reaches 200 mg m⁻² in the Ross Sea, while the concentration of chlorophyll a in the water column below is approximately 2 orders of magnitude less (47), and the percentage of metabolically active bacteria (32% [39]) is significantly higher than the 10% observed for temperate marine systems (36). The SIMCO is thus a major source of biomass in ice-covered regions of the Southern Ocean (59), providing a critical food source for grazing zooplankton (and, consequently, also for higher trophic levels) for much of the year (3, 59). This biomass is of particular importance during the darkness of the polar winter, where the bottom-ice community is the only available food source for juvenile krill. These grazers absolutely rely on the sea ice microbial community to survive, as the water lacks other food sources (6, 28).In the past decade, reports of the widespread occurrence of bacteriochlorophyll and PR pigments in planktonic marine bacteria have challenged the assumption that chlorophyll a is the only principal light-capturing pigment in ocean surface waters. These alternative pigments may in fact play a critical role in light energy harvesting for microbial metabolism in various aquatic ecosystems (5, 10, 25, 40, 49). It has been proposed that energy, rather than nutrient conservation, is important for the regulation of productivity (7). PR-containing phototrophic eubacteria could play a significant role in the energy budget of cells in the photic zone in marine environments (15). PR sequences have been detected in the Southern Ocean (9), but to our knowledge, there have been no reports of PR-bearing bacteria within the sea ice matrix.The majority of the microbial rhodopsin genes found in oceanic samples have been detected by environmental sequencing (30, 46, 48, 60). We have used degenerate PR gene primers (5) in this study to positively identify PR-bearing operational taxonomic units (OTUs) from sea ice. Also, specific bacterial mRNA can now be detected from extracted nucleic acids and used to examine gene expression and, thus, infer metabolic activity (8). With this in mind, we have generated cDNA from RNA extracted from sea ice samples. From these observations, we deduce that PR-bearing bacteria are present in sea ice and may be actively contributing to the ecosystem within this extreme microenvironment.     

Impacts of Inter- and Intralaboratory Variations on the Reproducibility of Microbial Community Analyses

With the advent of molecular biological techniques, especially next-generation sequencing and metagenomics, the number of microbial biogeography studies is rapidly increasing. However, these studies involve the synthesis of data generated by different laboratories using different protocols, chemicals, etc., all with inherent biases. The aim of this study was to assess inter- as well as intralaboratory variations in microbial community composition when standardized protocols are applied to a single soil sample. Aliquots from a homogenized soil sample from a rice field in Italy were sent to five participating laboratories. DNA was extracted by two investigators per laboratory using an identical protocol. All DNA samples were sent to one laboratory to perform DNA quantification, quantitative PCR (QPCR), and microarray and denaturing gradient gel electrophoresis (DGGE) analyses of methanotrophic communities. Yields, as well as purity of DNA, were significantly different between laboratories but in some cases also between investigators within the same laboratory. The differences in yield and quality of the extracted DNA were reflected in QPCR, microarray, and DGGE analysis results. Diversity indices (Shannon-Wiener, evenness, and richness) differed significantly between laboratories. The observed differences have implications for every project in which microbial communities are compared in different habitats, even if assessed within the same laboratory. To be able to make sensible comparisons leading to valid conclusions, intralaboratory variation should be assessed. Standardization of DNA extraction protocols and possible use of internal standards in interlaboratory comparisons may help in rendering a “quantifiable” bias.Microorganisms comprise a major part of total biomass and biodiversity (21, 41-43, 49). They play a critical role in biogeochemical processes and ecosystem functioning (16). However, knowledge of ecology and functioning of environmental microbial communities is still far from complete, mainly due to our inability to grow the majority of environmental microbes under laboratory conditions. The introduction of culture-independent DNA- and RNA-based techniques has led to a revolution in environmental microbiology, yielding a wealth of information on community compositions in an ever-growing range of habitats. Phylogenetic as well as functional microarrays (51) and metagenomic techniques (41, 47) enable in-depth analyses and comparison of whole microbial communities in a high-throughput manner.The collective goal of all environmental microbial ecology studies is 2-fold: (i) to obtain an overall understanding of microbial community composition, dynamics, and functioning and (ii) to identify regulating mechanisms. Reaching these goals will necessitate the integrated analyses of data generated in different laboratories and from different habitats. The first step in most if not all environmental microbial community studies is the extraction of total DNA from environmental samples in a way that reflects the in situ community composition as closely as possible. Numerous methods, protocols, and commercial kits have been developed to improve and optimize quantity and quality of extracted community DNA from a wide range of natural environments (4, 8, 28, 37, 39). However, up-to-date bias-free extraction methods are not available, especially not for complex and highly variable matrices, like soil. Beside the challenge of lysing all cells, the incomplete removal of compounds interfering with downstream processing render the development of a bias-free protocol a “mission impossible.” Assessments of the bias introduced by DNA extraction with different methods and kits on microbial community profiling revealed that a perfect protocol fitting all types of environments is not feasible (10, 17, 20, 45). However, in light of the global biodiversity debate, assessment of local and global patterns of microbial diversity and their controlling factors (19, 26) necessitates the comparison of data collected in multiple habitats and processed in different laboratories.In contrast to other scientific disciplines, intercalibration of protocols is not common practice in environmental microbiology. Interlaboratory comparisons (ring analyses) have been applied commonly in food control, veterinary, forensic, and soil studies to evaluate, for example, Salmonella diagnostic accuracy (25), virus isolation (18), enzyme-linked immmunosorbent assay methods (2), mitochondrial DNA sequencing (30), soil microbial biomass C (3), and quantitative PCR (QPCR) (11). Ring analyses assessing the reproducibility of DNA extraction and subsequent community analyses between different laboratories have not been carried out so far in environmental microbial ecology.A microbial functional guild that has been investigated intensively using molecular techniques is represented by the methanotrophs (aerobic methane-oxidizing bacteria [MOB]), which can be found in a wide variety of environments (27). The unique contribution of these bacteria to the global methane cycle has rendered the diversity and ecology of MOB hot topics for decades (9, 14, 34, 46, 48). By using methane as single source of carbon and energy, these microbes represent the only biological sink of the greenhouse gas methane under aerobic conditions (13). Aerobic MOB belong to the Gamma- and Alphaproteobacteria and the Verrucomicrobia (13, 34) and have the following features that enable linking function and identity. Assimilating methane facilitates the application of stable isotope probing of diagnostic lipids and of RNA/DNA (6, 29, 33). Besides this, the key gene in methane oxidation (for methanemonooxygenase subunit A, pmoA) reflects the phylogeny of these bacteria, facilitating a direct link between methane consumption and taxonomy. These features have made this group of microbes a model group for studies in environmental microbial ecology. Combined with the broad distribution and high environmental relevance, this group is highly suited to perform a ring analysis on reproducibility of DNA extraction and subsequent community profiling.In the present study, five independent laboratories from Norway, Finland, Netherlands, Germany, and Austria extracted DNA from the same rice field soil sample, using identical protocols and performed by two different investigators per laboratory. Subsequently, the extracted DNA was sent to one laboratory, where DNA quantification, QPCR, microarray, and denaturing gradient gel electrophoresis (DGGE) analyses were performed by one and the same person. The impacts of inter- as well as intralaboratory variations of DNA extraction are discussed, and recommendations for comparative studies are presented.     

Functional diversity and electron donor dependence of microbial populations capable of U(VI) reduction in radionuclide-contaminated subsurface sediments

In order to elucidate the potential mechanisms of U(VI) reduction for the optimization of bioremediation strategies, the structure-function relationships of microbial communities were investigated in microcosms of subsurface materials cocontaminated with radionuclides and nitrate. A polyphasic approach was used to assess the functional diversity of microbial populations likely to catalyze electron flow under conditions proposed for in situ uranium bioremediation. The addition of ethanol and glucose as supplemental electron donors stimulated microbial nitrate and Fe(III) reduction as the predominant terminal electron-accepting processes (TEAPs). U(VI), Fe(III), and sulfate reduction overlapped in the glucose treatment, whereas U(VI) reduction was concurrent with sulfate reduction but preceded Fe(III) reduction in the ethanol treatments. Phyllosilicate clays were shown to be the major source of Fe(III) for microbial respiration by using variable-temperature Mössbauer spectroscopy. Nitrate- and Fe(III)-reducing bacteria (FeRB) were abundant throughout the shifts in TEAPs observed in biostimulated microcosms and were affiliated with the genera Geobacter, Tolumonas, Clostridium, Arthrobacter, Dechloromonas, and Pseudomonas. Up to two orders of magnitude higher counts of FeRB and enhanced U(VI) removal were observed in ethanol-amended treatments compared to the results in glucose-amended treatments. Quantification of citrate synthase (gltA) levels demonstrated a stimulation of Geobacteraceae activity during metal reduction in carbon-amended microcosms, with the highest expression observed in the glucose treatment. Phylogenetic analysis indicated that the active FeRB share high sequence identity with Geobacteraceae members cultivated from contaminated subsurface environments. Our results show that the functional diversity of populations capable of U(VI) reduction is dependent upon the choice of electron donor.Uranium contamination in subsurface environments is a widespread problem at mining and milling sites across North America, South America, and Eastern Europe (1). Uranium in the oxidized state, U(VI), is highly soluble and toxic and thus is a potential contaminant to local drinking-water supplies (46). Nitrate is often a cocontaminant with U(VI) as a result of the use of nitric acid in the processing of uranium and uranium-bearing waste (6, 45). Oxidized uranium can be immobilized in contaminated groundwater through the reduction of U(VI) to insoluble U(IV) by indirect (abiotic) and direct (enzymatic) processes catalyzed by microorganisms. Current remediation practices favor the stimulation of reductive uranium immobilization catalyzed by indigenous microbial communities along with natural attenuation and monitoring (5, 24, 40, 44, 65, 68, 69). Microbial uranium reduction activity in contaminated subsurface environments is often limited by carbon or electron donor availability (13, 24, 44, 69). Previous studies have indicated that U(VI) reduction does not proceed until nitrate is depleted (13, 16, 24, 44, 68, 69), as high nitrate concentrations inhibit the reduction of U(VI) by serving as a competing and more energetically favorable terminal electron acceptor for microorganisms (11, 16). The fate and transport of uranium in groundwater are also strongly linked through sorption and precipitation processes to the bioreduction of Fe minerals, including oxides, layer-silicate clay minerals, and sulfides (7, 23, 53).In order to appropriately design U(VI) bioremediation strategies, the potential function and phylogenetic structure of indigenous subsurface microbial communities must be further understood (24, 34, 46). Conflicting evidence has been presented on which microbial groups, Fe(III)- or sulfate-reducing bacteria (FeRB or SRB), effectively catalyze the reductive immobilization of U(VI) in the presence of amended electron donors (5, 44, 69). The addition of acetate to the subsurface at a uranium-contaminated site in Rifle, Colorado, initially stimulated FeRB within the family Geobacteraceae to reduce U(VI) (5, 65). However, with long-term acetate addition, SRB within the family Desulfobacteraceae, which are not capable of U(VI) reduction, increased in abundance and a concomitant reoxidation of U(IV) was observed (5, 65). At a uranium-contaminated site in Oak Ridge, Tennessee, in situ and laboratory-based experiments successfully employed ethanol amendments to stimulate denitrification followed by the reduction of U(VI) by indigenous microbial communities (13, 24, 44, 48, 50, 57, 68). In these studies, ethanol amendments stimulated both SRB and FeRB, with SRB likely catalyzing the reduction of U(VI). This suggests that the potential for bioremediation will be affected by the choice of electron donor amendment through effects on the functional diversity of U(VI)-reducing microbial populations. As uranium reduction is dependent on the depletion of nitrate, the microbial populations mediating nitrate reduction are also critical to the design of bioremediation strategies. Although nitrate-reducing bacteria (NRB) have been studied extensively in subsurface environments (2, 15, 19, 24, 56, 58, 70), the mechanisms controlling the in situ metabolism of NRB remain poorly understood.The dynamics of microbial populations capable of U(VI) reduction in subsurface sediments are poorly understood, and the differences in the microbial community dynamics during bioremediation have not been explored. Based on the results of previous studies (13, 44, 49, 57, 68, 69), we hypothesized that the activity of nitrate- and Fe(III)-reducing microbial populations, catalyzing the reductive immobilization of U(VI) in subsurface radionuclide-contaminated sediments, would be dependent on the choice of electron donor. The objectives of the present study were (i) to characterize structure-function relationships for microbial groups likely to catalyze or limit U(VI) reduction in radionuclide-contaminated sediments and (ii) to further develop a proxy for the metabolic activity of FeRB. Microbial activity was assessed by monitoring terminal electron-accepting processes (TEAPs), electron donor utilization, and Fe(III) mineral transformations in microcosms conducted with subsurface materials cocontaminated with high levels of U(VI) and nitrate. In parallel, microbial functional groups (i.e., NRB and FeRB) were enumerated and characterized using a combination of cultivation-dependent and -independent methods.     

Use of Ichip for High-Throughput In Situ Cultivation of “Uncultivable” Microbial Species

One of the oldest unresolved microbiological phenomena is why only a small fraction of the diverse microbiological population grows on artificial media. The “uncultivable” microbial majority arguably represents our planet''s largest unexplored pool of biological and chemical novelty. Previously we showed that species from this pool could be grown inside diffusion chambers incubated in situ, likely because diffusion provides microorganisms with their naturally occurring growth factors. Here we utilize this approach and develop a novel high-throughput platform for parallel cultivation and isolation of previously uncultivated microbial species from a variety of environments. We have designed and tested an isolation chip (ichip) composed of several hundred miniature diffusion chambers, each inoculated with a single environmental cell. We show that microbial recovery in the ichip exceeds manyfold that afforded by standard cultivation, and the grown species are of significant phylogenetic novelty. The new method allows access to a large and diverse array of previously inaccessible microorganisms and is well suited for both fundamental and applied research.It has been known for over a century that the overwhelming majority of microbial species do not grow on synthetic media in vitro and remain unexplored (13, 32, 37, 39, 40, 43). The rRNA and metagenomics approaches demonstrated a spectacular diversity of these uncultivated species (11, 21, 25-27, 30, 36). Accessing this “missing” microbial diversity is of significant interest for both basic and applied sciences and has been recognized as one of the principal challenges for microbiology today (12, 29, 41). In recent years, technical advances in cultivation methodologies have recovered a diverse set of ecologically relevant species (1, 3, 5, 7, 15, 20, 24, 28, 33, 42). However, by and large the gap between microbial diversity in nature and that in culture collections remains unchanged, and most microbial phyla still have no cultivable representatives (25, 29). Earlier, we developed a novel method of in situ cultivation of environmental microorganisms inside diffusion chambers (15). The rationale for such an approach was that diffusion would provide cells inside the chamber with naturally occurring growth components and enable those species that grew in nature at the time of the experiment to also grow inside the diffusion chambers. Expectedly, this method yields a rate of microbial recovery many times larger than those of standard techniques. Even so, this method is laborious and does not allow an efficient, high-throughput isolation of microbial species en masse. This limits the method''s applicability, for example, in the drug discovery effort. Here we transform this methodology into a high-throughput technology platform for massively parallel cultivation of “uncultivable” species. Capitalizing on earlier microfluidics methods developed for microbial storage and screening (4, 16), we have designed and tested an isolation chip, or ichip for short, which consists of hundreds of miniature diffusion chambers. If each diffusion minichamber is loaded with a single cell, the resulting culture is monospecific. The ichip thus allows microbial growth and isolation into pure culture in one step. Here we demonstrate that cultivation of environmental microorganisms inside the ichip incubated in situ leads to a significantly increased colony count over that observed on synthetic media. Perhaps even more significantly, species grown in ichips are different from those registered in standard petri dishes and are highly novel.     

Interactions of DNA with Biofilm-Derived Membrane Vesicles

The biofilm matrix contributes to the chemistry, structure, and function of biofilms. Biofilm-derived membrane vesicles (MVs) and DNA, both matrix components, demonstrated concentration-, pH-, and cation-dependent interactions. Furthermore, MV-DNA association influenced MV surface properties. This bears consequences for the reactivity and availability for interaction of matrix polymers and other constituents.The biofilm matrix contributes to the chemistry, structure, and function of biofilms and is crucial for the development of fundamental biofilm properties (46, 47). Early studies defined polysaccharides as the matrix component, but proteins, lipids, and nucleic acids are all now acknowledged as important contributors (7, 15). Indeed, DNA has emerged as a vital participant, fulfilling structural and functional roles (1, 5, 6, 19, 31, 34, 36, 41, 43, 44). The phosphodiester bond of DNA renders this polyanionic at a physiological pH, undoubtedly contributing to interactions with cations, humic substances, fine-dispersed minerals, and matrix entities (25, 41, 49).In addition to particulates such as flagella and pili, membrane vesicles (MVs) are also found within the matrices of gram-negative and mixed biofilms (3, 16, 40). MVs are multifunctional bilayered structures that bleb from the outer membranes of gram-negative bacteria (reviewed in references 4, 24, 27, 28, and 30) and are chemically heterogeneous, combining the known chemistries of the biofilm matrix. Examination of biofilm samples by transmission electron microscopy (TEM) has suggested that matrix material interacts with MVs (Fig. ​(Fig.1).1). Since MVs produced in planktonic culture have associated DNA (11, 12, 13, 20, 21, 30, 39, 48), could biofilm-derived MVs incorporate DNA (1, 39, 40, 44)?Open in a separate windowFIG. 1.Possible interactions between matrix polymers and particulate structures. Shown is an electron micrograph of a thin section through a P. aeruginosa PAO1 biofilm. During processing, some dehydration occurred, resulting in collapse of matrix material into fibrillate arrangements (black filled arrows). There is a suggestion of interactions occurring with particulate structures such as MVs (hollow white arrow) and flagella (filled white arrows) (identified by the appearance and cross-dimension of these highly ordered structures when viewed at high magnification), which was consistently observed with other embedded samples and also with whole-mount preparations of gently disrupted biofilms (data not shown). The scale bar represents 200 nm.     

Strategy for Extracting DNA from Clay Soil and Detecting a Specific Target Sequence via Selective Enrichment and Real-Time (Quantitative) PCR Amplification

We present a simple strategy for isolating and accurately enumerating target DNA from high-clay-content soils: desorption with buffers, an optional magnetic capture hybridization step, and quantitation via real-time PCR. With the developed technique, μg quantities of DNA were extracted from mg samples of pure kaolinite and a field clay soil.Isolating and characterizing DNA sequences for use in molecular methods are integral to evaluating microbial community diversity in soil (6, 21, 22, 24, 37). Any isolation protocol should maximize nucleic acid isolation while minimizing copurification of enzymatic inhibitors. Although several methods that focus on extraction of total community DNA from environmental soil and water samples have been published (7, 21, 26, 34), the lack of a standard nucleic acid isolation protocol (32) reflects the difficulty in accomplishing these goals, most likely due to the complex nature of the soil environment.DNA extraction is especially difficult for soils containing clay (3, 5), given the tight binding of DNA strands to clay soil particles (7, 10, 20). Additionally, extracellular DNA binds to and is copurified with soil humic substances (10), which inhibit the activity of enzymes such as restriction endonucleases and DNA polymerase (6, 13, 23). Although clay-bound DNA can be PCR amplified in the absence of inhibitors (1), it is often the case that inhibitors are present in the soil environment, among them bilirubin, bile salts, urobilinogens, and polysaccharides (40). Of these inhibitors, humic substances have been found to be the most recalcitrant (36).A promising technique for isolating specific target sequences from soil particles and enzymatic inhibitors is the magnetic capture hybridization-PCR technique (MCH-PCR) presented by Jacobsen (19) and used to obtain high detection sensitivities (11, 38).We have found no evidence in the published literature of the use of MCH-PCR on soils that have high clay contents and here present a three-step strategy for isolating specific DNA sequences from the most difficult soil environment—clay that contains humic substances—and enumerating a specific target sequence from the crude extract.     

Poly-N-Acetylglucosamine Matrix Polysaccharide Impedes Fluid Convection and Transport of the Cationic Surfactant Cetylpyridinium Chloride through Bacterial Biofilms

Biofilms are composed of bacterial cells encased in a self-synthesized, extracellular polymeric matrix. Poly-β(1,6)-N-acetyl-d-glucosamine (PNAG) is a major biofilm matrix component in phylogenetically diverse bacteria. In this study we investigated the physical and chemical properties of the PNAG matrix in biofilms produced in vitro by the gram-negative porcine respiratory pathogen Actinobacillus pleuropneumoniae and the gram-positive device-associated pathogen Staphylococcus epidermidis. The effect of PNAG on bulk fluid flow was determined by measuring the rate of fluid convection through biofilms cultured in centrifugal filter devices. The rate of fluid convection was significantly higher in biofilms cultured in the presence of the PNAG-degrading enzyme dispersin B than in biofilms cultured without the enzyme, indicating that PNAG decreases bulk fluid flow. PNAG also blocked transport of the quaternary ammonium compound cetylpyridinium chloride (CPC) through the biofilms. Binding of CPC to biofilms further impeded fluid convection and blocked transport of the azo dye Allura red. Bioactive CPC was efficiently eluted from biofilms by treatment with 1 M sodium chloride. Taken together, these findings suggest that CPC reacts directly with the PNAG matrix and alters its physical and chemical properties. Our results indicate that PNAG plays an important role in controlling the physiological state of biofilms and may contribute to additional biofilm-associated processes such as biocide resistance.Biofilms are composed of bacterial cells encased in a self-synthesized, extracellular polymeric matrix (7). The main function of the biofilm matrix is to provide a structural framework that holds the cells together in a mass and firmly attaches the bacterial mass to the underlying surface. In addition to having a structural role, the matrix provides biofilm cells with a protected microenvironment containing dissolved nutrients, secreted enzymes, DNA, and phages. The matrix may also contribute to the increased antimicrobial resistance exhibited by biofilm cells, either by providing a diffusion barrier or by directly binding to antimicrobial agents and preventing their penetration into the biofilm (19).Polysaccharides are a major matrix component in most bacterial biofilms (26). Poly-β(1,6)-N-acetyl-d-glucosamine (PNAG) is an extracellular polysaccharide that mediates biofilm cohesion in numerous gram-negative members of the Proteobacteria family, including Escherichia coli, Yersinia pestis, Pseudomonas fluorescens, Bordetella spp., Xenorhabdus nematophila, Aggregatibacter actinomycetemcomitans, and Actinobacillus pleuropneumoniae (4, 8, 15, 22), and in the gram-positive species Staphylococcus aureus and Staphylococcus epidermidis (3, 17). Specific biofilm-related functions ascribed to PNAG include abiotic surface attachment (1), epithelial cell attachment (23, 28), intercellular adhesion (15, 17), and resistance to killing by antibiotics, detergents, antimicrobial peptides, and mammalian phagocytic cells (9, 10, 16, 27, 29).In the present study we investigated the physical and chemical properties of the PNAG matrix in biofilms produced by the porcine respiratory pathogen A. pleuropneumoniae and the device-associated pathogen S. epidermidis. By using a novel centrifugal filter device assay, we obtained evidence that PNAG significantly inhibits fluid convection and solute transport through A. pleuropneumoniae and S. epidermidis biofilms.