Unattached Bacteria Research Articles

Functional diversity of bacterial microbiota associated with the toxigenic benthic dinoflagellate Prorocentrum.

Interactions between bacterial microbiota and epibenthic species of the dinoflagellate Prorocentrum may define the onset and persistence of benthic harmful algal blooms (bHABs). Chemical ecological interactions within the dinoflagellate phycosphere potentially involve a complex variety of organic molecules, metabolites, and toxins, including undefined bioactive compounds. In this study, the bacterial diversity and core members of the dinoflagellate-associated microbiota were defined from 11 strains of three epibenthic Prorocentrum species, representing three geographically disjunct locations within Mexican coastal waters. Microbiota profiles in stable monoclonal Prorocentrum cultures were obtained by sequencing amplicons of the V3-V4 region of the 16S rRNA gene. Thirteen classes of bacteria were identified among dinoflagellate clones, where Alphaproteobacteria, Gammaproteobacteria, and Bacteroidia were consistently dominant. The bacterial community structure exhibited significantly different grouping by the location of origin of dinoflagellate clones. No significant diversity difference was found among free-living or unattached bacteria in the dinoflagellate culture medium (M) compared with those in closer association with the dinoflagellate host cells (H). Twelve taxa were defined as core members of the bacterial assemblage, representing the genera Algiphilus, Cohaesibacter, Labrenzia, Mameliella, Marinobacter, Marivita, Massilia, Muricauda, Roseitalea, and an unclassified member of the Rhodobacteraceae. The core members are inferred to significantly contribute to primary and secondary metabolic functions, but no direct correlation with dinoflagellate toxigenicity was apparent. Overall the bacterial profile and implied gene functionality indicated a suite of positive interactions, suggesting either mutualism or commensalism with the dinoflagellate. The further characterization and interpretation of specific gene functions and interactions between bacteria and dinoflagellates, such as epibenthic members of genus Prorocentrum, are key to understanding their role in toxigenesis and bHAB development.

Bacterial Adhesion Kinetics in a High Throughput Setting in Seconds-minutes Time Resolution.

Bacterial surface adhesion, the first step in many important processes including biofilm formation and tissue invasion, is a fast process that occurs on a time scale of seconds. Adhesion patterns tend to be stochastic and spatially heterogeneous, especially when bacteria are present in low population densities and at early stages of adhesion to the surface. Thus, in order to observe this process, a high degree of temporal resolution is needed across a large surface area in a way that allows several replicates to be monitored. Some of the current methods used to measure bacterial adhesion include microscopy, staining-based microtiter assays, spectroscopy, and PCR. Each of these methods has advantages in assaying aspects of bacterial surface adhesion, but none can capture all features of the process. In the protocol presented here, adapted from Shteindel et al., 2019 , fluorescently-labeled bacteria are monitored in a multi-titer setting using a standard plate fluorimeter and a dye that absorbs light in the fluorophore excitation and emission wavelengths. The advantage of using this dye is that it restricts the depth of the optic layer to the few microns adjacent to the bottom of the microtiter well, eliminating fluorescence originating from unattached bacteria. Another advantage of this method is that this setting does not require any preparatory steps, which enables reading of the sample to be repeated or continuous. The use of a standard multi-titer well allows easy manipulation and provides flexibility in experimental design.

Relationship between aquifer biofilms and unattached microbial indicators of urban groundwater contamination.

Aquifers, springs and other groundwater-dependent ecosystems are threatened by urban land use, which causes water quality deterioration through nutrient loading, sewage infiltration, groundwater extraction and, along coasts, seawater intrusion. The presence of certain microbes in groundwater can indicate that an aquifer is anthropogenically contaminated. Interpretations made from observations of indicator microbes in groundwater are limited because the relationship between the presumably allochthonous indicator microbes and relevant autochthonous microbial communities has not been characterized. This study addressed whether autochthonous aquifer biofilms can influence the presence of presumed microbial indicators in groundwater, and simultaneously used microbial indicators to trace sources of urban contamination at a karst spring of conservation concern. These questions were approached using a 17-month time series analysis of attached biofilm and adjacent unattached bacteria in the submerged karst aquifer conduit associated with this spring. Environmental 16S rRNA gene sequencing was performed to characterize these communities, and community structure data were contextualized with groundwater geochemical and hydrogeological measurements. Linear regression models were developed to explain the relative abundance patterns of indicator microbes and other unattached microbes at this site. The results of this study suggest that dominant aquifer biofilms do not influence the presence of unattached microbial taxa that are presumed to be indicators of groundwater contamination, and generated new information about the origin of coliform bacteria at the study site. These results build confidence in the use of microbial indicators in groundwater-dependent ecosystem conservation strategies and inform future management plans for urban aquifers and springs worldwide.

Novel and known periodontal pathogens residing in gingival crevicular fluid are associated with rheumatoid arthritis.

BackgroundPeriodontitis is a suspected environmental risk factor for the development of rheumatoid arthritis (RA). However, correlation mechanisms between the two pathologies remain elusive. This study examined potential correlations between detached subgingival bacteria collected in gingival crevicular fluid (GCF) and RA parameters.MethodsRA patients (n = 52, F:M = 40:12), patients with Behcet's disease (BD, n = 40, F:M = 29:11) as another systemic inflammatory disease were studied along with a systemically healthy control group (HC, n = 57, F:M = 40:17). All participants were non‐smokers. Full mouth periodontal parameters were recorded. RA activity was assessed using the 28‐joint Disease Activity Score (DAS‐28). Rheumatoid factors (RFs)‐IgM and ‐IgA were measured by ELISA. GCF samples were investigated by means of fluorescent in situ hybridization for 10 different bacterial taxa.ResultsThe taxa TM7, Synergistetes cluster B, Leptotrichia, Megasphaera, Anaeroglobus geminatus, and Tannerella forsythia displayed significantly differential abundances between the groups. Whereas abundances of Megasphaera and A. geminatus were significantly increased in the RA group, only Porphyromonas gingivalis displayed significant correlations with plaque scores, bleeding on probing, and RF‐IgA. RA patients displaying RF‐IgA levels >75 IU/mL exhibited five‐fold more abundant P. gingivalis levels than patients below the threshold. This association with RF‐IgA levels appeared even more pronounced, by six‐fold more P. gingivalis (P = 0.025), in patients with a DAS‐28 score >3.2, indicative of moderate/very active RA.ConclusionsUnattached GCF bacteria may mediate the association between periodontitis and RA, and monitoring the bacterial composition of GCF might inform on RA activity. The role of newly identified bacterial taxa in RA warrants further investigations.

Vancomycin-loaded electrospun polycaprolactone/nano-hydroxyapatite membrane for the treatment of blood infections

Nowadays, because of the resistance of bacteria to antibiotics, researchers are trying to make new antibiotics or sometimes even bring them back into the treatment cycle so that they could eliminate the bacteria’s resistance. On the other hand, the use of nanofibers has become widespread in many fields for their unique properties and convenient design. The present study focuses on the production of hydrophobic nanofibers to absorb the bacteria and their toxins from the bloodstream that contains the infection. Many bacterial surfaces have hydrophobic surfactant properties due to hydrophobic surface protein. According to the principle of binding two hydrophobic molecules to each other in an aqueous medium, the nanofibers are designed to physically absorb the bacteria. The use of antibiotics in the study can remove some unattached bacteria. In addition, using nanofiber manufacturing techniques can reduce the resistance of bacteria to antibiotics. The construction of the desired membrane can be used in subsequent studies as a replacement membrane for dialysis filters.

Pseudomonas aeruginosa cells attached to a surface display a typical proteome early as 20 minutes of incubation.

Biofilms are present in all environments and often result in negative effects due to properties of the biofilm lifestyle and especially antibiotics resistance. Biofilms are associated with chronic infections. Controlling bacterial attachment, the first step of biofilm formation, is crucial for fighting against biofilm and subsequently preventing the persistence of infection. Thus deciphering the underlying molecular mechanisms involved in attachment could allow discovering molecular targets from it would be possible to develop inhibitors against bacterial colonization and potentiate antibiotherapy. To identify the key components and pathways that aid the opportunistic pathogen Pseudomonas aeruginosa in attachment we performed for the first time a proteomic analysis as early as after 20 minutes of incubation using glass wool fibers as a surface. We compared the protein contents of the attached and unattached bacteria. Using mass spectrometry, 3043 proteins were identified. Our results showed that, as of 20 minutes of incubation, using stringent quantification criteria 616 proteins presented a modification of their abundance in the attached cells compared to their unattached counterparts. The attached cells presented an overall reduced gene expression and characteristics of slow-growing cells. The over-accumulation of outer membrane proteins, periplasmic folding proteins and O-antigen chain length regulators was also observed, indicating a profound modification of the cell envelope. Consistently the sigma factor AlgU required for cell envelope homeostasis was highly over-accumulated in attached cells. In addition our data suggested a role of alarmone (p)ppGpp and polyphosphate during the early attachment phase. Furthermore, almost 150 proteins of unknown function were differentially accumulated in the attached cells. Our proteomic analysis revealed the existence of distinctive biological features in attached cells as early as 20 minutes of incubation. Analysis of some mutants demonstrated the interest of this proteomic approach in identifying genes involved in the early phase of adhesion to a surface.

The contribution of marine aggregate-associated bacteria to the accumulation of pathogenic bacteria in oysters: an agent-based model.

Bivalves process large volumes of water, leading to their accumulation of bacteria, including potential human pathogens (e.g., vibrios). These bacteria are captured at low efficiencies when freely suspended in the water column, but they also attach to marine aggregates, which are captured with near 100% efficiency. For this reason, and because they are often enriched with heterotrophic bacteria, marine aggregates have been hypothesized to function as important transporters of bacteria into bivalves. The relative contribution of aggregates and unattached bacteria to the accumulation of these cells, however, is unknown. We developed an agent‐based model to simulate accumulation of vibrio‐type bacteria in oysters. Simulations were conducted over a realistic range of concentrations of bacteria and aggregates and incorporated the dependence of pseudofeces production on particulate matter. The model shows that the contribution of aggregate‐attached bacteria depends strongly on the unattached bacteria, which form the colonization pool for aggregates and are directly captured by the simulated oysters. The concentration of aggregates is also important, but its effect depends on the concentration of unattached bacteria. At high bacterial concentrations, aggregates contribute the majority of bacteria in the oysters. At low concentrations of unattached bacteria, aggregates have a neutral or even a slightly negative effect on bacterial accumulation. These results provide the first evidence suggesting that the concentration of aggregates could influence uptake of pathogenic bacteria in bivalves and show that the tendency of a bacterial species to remain attached to aggregates is a key factor for understanding species‐specific accumulation.

The Vitamin B1 and B12 Required by the Marine Dinoflagellate Lingulodinium polyedrum Can be Provided by its Associated Bacterial Community in Culture.

In this study we established the B1 and B12 vitamin requirement of the dinoflagellate Lingulodinium polyedrum and the vitamin supply by its associated bacterial community. In previous field studies the B1 and B12 demand of this species was suggested but not experimentally verified. When the axenic vitamin un-supplemented culture (B-ns) of L. polyedrum was inoculated with a coastal bacterial community, the dinoflagellate’s vitamin growth limitation was overcome, reaching the same growth rates as the culture growing in vitamin B1B7B12-supplemented (B-s) medium. Measured B12 concentrations in the B-s and B-ns cultures were both higher than typical coastal concentrations and B12 in the B-s culture was higher than in the B-ns culture. In both B-s and B-ns cultures, the probability of dinoflagellate cells having bacteria attached to the cell surface was similar and in both cultures an average of six bacteria were attached to each dinoflagellate cell. In the B-ns culture the free bacterial community showed significantly higher cell abundance suggesting that unattached bacteria supplied the vitamins. The fluorescence in situ hybridization (FISH) protocol allowed the quantification and identification of three bacterial groups in the same samples of the free and attached epibiotic bacteria for both treatments. The relative composition of these groups was not significantly different and was dominated by Alphaproteobacteria (>89%). To complement the FISH counts, 16S rDNA sequencing targeting the V3–V4 regions was performed using Illumina-MiSeq technology. For both vitamin amendments, the dominant group found was Alphaproteobacteria similar to FISH, but the percentage of Alphaproteobacteria varied between 50 and 95%. Alphaproteobacteria were mainly represented by Marivita sp., a member of the Roseobacter clade, followed by the Gammaproteobacterium Marinobacter flavimaris. Our results show that L. polyedrum is a B1 and B12 auxotroph, and acquire both vitamins from the associated bacterial community in sufficient quantity to sustain the maximum growth rate.

Influence of wave action on the partitioning and transport of unattached and floc-associated bacteria in fresh water.

The dynamic interaction of bacteria within bed sediment and suspended sediment (i.e., floc) in a wave-dominated beach environment was assessed using a laboratory wave flume. The influence of shear stress (wave energy) on bacterial concentrations and on the partitioning and transport of unattached and floc-associated bacteria was investigated. The study showed that increasing wave energy (0.60 and 5.35 N/s) resulted in a 0.5 to 1.5 log increase in unattached cells of the test bacterium Pseudomonas sp. strain CTO7::gfp-2 in the water column. There was a positive correlation between the bacterial concentrations in water and the total suspended solids, with the latter increasing from values of near 0 to up to 200 mg/L over the same wave energy increase. The median equivalent spherical diameter of flocs in suspension also increased by an order of magnitude in all experimental trials. Under both low (0.60 N/s) and high (5.35 N/s) energy regime, bacteria were shown to preferentially associate with flocs upon cessation of wave activity. The results suggest that collecting water samples during periods of low wave action for the purpose of monitoring the microbiological quality of water may underestimate bacterial concentrations partly because of an inability to account for the effect of shear stress on the erosion and mobilization of bacteria from bed sediment to the water column. This highlights the need to develop a more comprehensive beach analysis strategy that not only addresses presently uncharacterized shores and sediments but also recognizes the importance of eroded flocs as a vector for the transport of bacteria in aquatic environments.

Influence of PVM/MA copolymer on bacterial adherence to resin surfaces

Purpose: The aim of this pilot study was to investigate the effects of methylvinyl ether and maleic acid (PVM/MA) copolymer containing self-etch (SE) adhesive bonding agent in preventing colonization of the composite material surfaces by Streptococcus gordonii. Methods and materials: Twelve polished cylindrical composite resin discs (8mm in diameter and 2mm in thickness) were divided into two test groups: 1. SE adhesive resin (Clearfil SE Bond, Kuraray) application to the top and bottom surfaces of the discs; 2. PVM/MA containing SE adhesive resin application. For PVM/MA containing adhesive resin, 3mg PVM/MA powder was mixed in 2 drops of bonding agent of SE Bond adhesive material. The specimens were placed in 24-well microplates and sterilized by UV irradiation for 30min before incubation with bacteria. Bacterial adherence to composite surfaces was assessed by determining colony-forming units (CFU) and viewing by scanning electron microscopy (SEM). The specimens were immersed in clear and filtered whole saliva which had been collected from a single donor. After washing with phosphate-buffered saline (PBS), the specimens were placed in individual wells containing 1× 108 cells/ml of Streptococcus gordonii in PBS. Immersed specimens were incubated at 37 ◦C for 24h and washed again with PBS to remove unattached bacteria. Half of the specimens were then placed into tubes containing 1mL of PBS. The attached bacterial cells were dispersed by sonication. Serial dilutions were made and inoculated ontoBrainHeart InfusionBrothplates to determine CFU. For SEM analysis, the other half of the specimens were immersed in Karnovsky’s fixative for 24h, dehydrated, coated with gold-palladium. ANOVA and post hoc Tukey’s test were used to assess statistical differences. Results: Bacterial adherence (log CFU/mL) to PVM/MA containing SE adhesive resin-covered composite surfaces was significantly less (9.20E+02) than adherence (7.92E+03) to regular SE adhesive resin covered surfaces (p=0.05). SEM also confirmed these results showing reduced bacterial attachment to PVM/MA containing adhesive resin applied surfaces. Conclusion: The PVM/MA copolymer significantly reduces bacterial adherence to resin materials.

Investigation of Mercury Methylation Pathways in Biofilm versus Planktonic Cultures of Desulfovibrio desulfuricans

Biofilms can methylate mercury (Hg) at higher rates than unattached bacteria and are increasingly recognized as important Hg methylation sites in the environment. Our previous study showed that methylation rates in biofilm cultures were up to 1 order of magnitude greater than those in planktonic cultures of a sulfate-reducing bacterium. To probe whether the differential Hg methylation rates resulted from metabolic differences between these two cultures, Hg methylation assays following molybdate or chloroform inhibition (a specific inhibitor of the acetyl-CoA pathway) were conducted on biofilm and planktonic cultures of Desulfovibrio desulfuricans strains M8 and ND132. Molybdate was as effective in inhibiting Hg methylation as well as growth in both planktonic and biofilm cultures. The addition of chloroform only impacted Hg methylation in biofilm cultures, suggesting that different pathways are used for methylation in biofilm compared to planktonic cultures. To investigate this further, expression of the cooS gene, which encodes for carbon monoxide dehydrogenase, a key enzyme in the acetyl-CoA pathway, was compared in biofilm and planktonic cultures of ND132. Biofilm cultures showed up to 4 times higher expression of cooS than planktonic cultures. On the basis of these results, the acetyl-CoA pathway appears to play an important role in methylation in biofilm cultures of this organism, possibly by supplying the methyl group to Hg methylating enzymes; methylation in planktonic cultures appears to be independent of this pathway. This observation has important implications, particularly in developing reliable models to predict Hg methylation rates in different environments and perhaps eventually in being able to control this undesirable chemical transformation.

Analysis of the Attached Microbial Community on Mucilaginous Cyanobacterial Aggregates in the Eutrophic Lake Taihu Reveals the Importance of Planctomycetes

The phylogenetic diversity of the microbial community assemblage of the carpet-like mucilaginous cyanobacterial blooms in the eutrophic Lake Taihu was investigated. 16S ribosomal DNA clone libraries produced from the DNA of cyanobacterial assemblages that had been washed to remove unattached bacteria contained only cyanobacteria. However, a further treatment which included grinding the freeze-dried material to physically detach cells followed by the removal of larger cells by filtration allowed us to detect a large variety of bacteria within the cyanobacterial bloom community. Interestingly, the dominant members of the microbial community were Planctomycetes followed by Cytophaga-Flavobacterium-Bacteroides (CFB), Betaproteobacteria, and Gammaproteobacteria. The analysis of the 16S ribosomal DNA clone libraries made from enrichment culture revealed much higher phylogenetic diversity of bacteria. Dominant bacterial groups in the enrichment system were identified as members of the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria subdivisions, CFB group, and Planctomycetes. In addition, the clone libraries constructed from Planctomycetes-specific 16S ribosomal RNA primers also verified that the enrichment allowed a diversity of Planctomycetes to proliferate, although the community composition was altered after enrichment.

Autonomous bacterial localization and gene expression based on nearby cell receptor density

Escherichia coli were genetically modified to enable programmed motility, sensing, and actuation based on the density of features on nearby surfaces. Then, based on calculated feature density, these cells expressed marker proteins to indicate phenotypic response. Specifically, site-specific synthesis of bacterial quorum sensing autoinducer-2 (AI-2) is used to initiate and recruit motile cells. In our model system, we rewired E. coli's AI-2 signaling pathway to direct bacteria to a squamous cancer cell line of head and neck (SCCHN), where they initiate synthesis of a reporter (drug surrogate) based on a threshold density of epidermal growth factor receptor (EGFR). This represents a new type of controller for targeted drug delivery as actuation (synthesis and delivery) depends on a receptor density marking the diseased cell. The ability to survey local surfaces and initiate gene expression based on feature density represents a new area-based switch in synthetic biology that will find use beyond the proposed cancer model here.

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37°C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 μl tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37°C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 μl tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

Specified Species in Gingival Crevicular Fluid Predict Bacterial Diversity

BackgroundAnalysis of gingival crevicular fluid (GCF) samples may give information of unattached (planktonic) subgingival bacteria. Our study represents the first one targeting the identity of bacteria in GCF.Methodology/Principal FindingsWe determined bacterial species diversity in GCF samples of a group of periodontitis patients and delineated contributing bacterial and host-associated factors. Subgingival paper point (PP) samples from the same sites were taken for comparison. After DNA extraction, 16S rRNA genes were PCR amplified and DNA-DNA hybridization was performed using a microarray for over 300 bacterial species or groups. Altogether 133 species from 41 genera and 8 phyla were detected with 9 to 62 and 18 to 64 species in GCF and PP samples, respectively, per patient. Projection to latent structures by means of partial least squares (PLS) was applied to the multivariate data analysis. PLS regression analysis showed that species of genera including Campylobacter, Selenomonas, Porphyromonas, Catonella, Tannerella, Dialister, Peptostreptococcus, Streptococcus and Eubacterium had significant positive correlations and the number of teeth with low-grade attachment loss a significant negative correlation to species diversity in GCF samples. OPLS/O2PLS discriminant analysis revealed significant positive correlations to GCF sample group membership for species of genera Campylobacter, Leptotrichia, Prevotella, Dialister, Tannerella, Haemophilus, Fusobacterium, Eubacterium, and Actinomyces.Conclusions/SignificanceAmong a variety of detected species those traditionally classified as Gram-negative anaerobes growing in mature subgingival biofilms were the main predictors for species diversity in GCF samples as well as responsible for distinguishing GCF samples from PP samples. GCF bacteria may provide new prospects for studying dynamic properties of subgingival biofilms.

Evaluation of bacteria‐facilitated cadmium transport in gravel columns using the HYDRUS colloid‐facilitated solute transport model

The colloid‐facilitated solute transport model, based on HYDRUS‐1D, was evaluated using the column experimental data of Pang et al. (2005) for cadmium (Cd) transport facilitated by B. subtilis spores or E. coli in saturated coarse alluvial gravels. We simulated Cd transport involving convection, dispersion, kinetic adsorption/desorption to/from the aquifer media and to/from mobile/immobile bacteria, and kinetic attachment/detachment of the bacteria to/from the aquifer media. To reduce the number of parameters to be optimized, we independently estimated Cd sorption/desorption rates to mobile bacteria from a batch study. The model described the collected experimental data reasonably well. Extensive sensitivity analysis to various reaction parameters was carried out to obtain an understanding of the relative importance of individual model parameters on model predictions. Our modeling results suggest that the rates of Cd sorption or desorption differ not only between different bacterial species but also between unattached and deposited bacteria. The results of the sensitivity analysis indicated that the Cd sorption rate to unattached bacteria had a significantly greater impact on the model results than its sorption rate to deposited bacteria. For the experimental system investigated here, model results were most sensitive to parameters describing interactions between Cd‐aquifer media, bacteria‐aquifer media, and Cd‐mobile bacteria, and they were less sensitive to interactions between Cd‐immobile bacteria and desorption rate from mobile bacteria.

Identification of oral bacteria associated with crevicular epithelial cells from chronic periodontitis lesions

Bacterial invasion of host epithelial cells plays an important role in the pathogenesis of periodontal diseases; however, the interactions between subgingival species and the gingival crevice cells are not fully understood. This study determined the prevalence of a group of oral bacterial species on or in epithelial cells derived from periodontal pockets and the gingival crevice of subjects with periodontitis. Samples of epithelial cells were obtained from 120 sites with periodontal pockets > or =4 mm and 92 periodontally healthy sites from 49 patients (mean age 46.3+/-1.4 years; 43% males) with chronic periodontitis. Bacteria in or on epithelial cells were separated from unattached bacteria by Percoll density-gradient centrifugation. The presence and levels of 33 oral species were determined in epithelial cell samples by whole genomic DNA probes and the checkerboard method. The most frequently detected species were Porphyromonas gingivalis (42%), Treponema denticola (38%), Prevotella intermedia (37%), Streptococcus intermedius (36%), Campylobacter rectus (35%), Streptococcus sanguinis (35%) and Streptococcus oralis (34%). Species of Actinomyces were found in low prevalence and levels. The data indicated that there were more micro-organisms on or in epithelial cells obtained from periodontal pockets than from healthy sulci; however, no significant differences regarding the percentage and level of any specific species were found between these sites. Veillonella parvula, S. oralis, Streptococcus gordonii and Streptococcus mitis tended to be more prevalent in sites without disease. These findings demonstrated that a wide range of oral species may be detected on or in crevicular epithelial cells from sites with periodontitis and from periodontally healthy sulci.

Field evidence for a protistan role in an organically-contaminated aquifer.

The association between protists, bacteria, and dissolved organic carbon (DOC) in an oxygen-depleted, 6 km-long wastewater contaminant plume within a sandy aquifer (Cape Cod, MA) was investigated by comparing abundance patterns along longitudinal and vertical transects and at a control site. Strong linear correlations were observed between unattached bacterial abundance and DOC for much of the upgradient-half of the plume (0.1-2.5 km downgradient from the source) that is characterized by quasi-steady state chemistry. However, a logarithmic decrease was observed between the number of protists supported per mg of DOC and the estimated age of the DOC within the plume. The relatively labile dissolved organic contaminants that characterize the groundwater sampled from the plume < or = 0.1 km downgradient from the contaminant source appeared to indirectly support 3-4 times as many protists (per mg of DOC) as the older, more recalcitrant DOC in the alkylbenzene sulfonate (ABS)-contaminated zone at 3 km downgradient (approximately 30 years travel time). Substantive numbers of protists (>10(4)/cm3) were recovered from suboxic zones of the plume. The higher than expected ratios of protists to unattached bacteria (10 to 100:1) observed in much of the plume suggest that protists may be grazing upon both surface-associated and unattached bacterial communities to meet their nutritional requirements. In closed bottle incubation experiments, the presence of protists caused an increase in bacterial growth rate, which became more apparent at higher amendments of labile DOC (3-20 mgC/L). The presence of protists resulted in an increase in the apparent substrate saturation level for the unattached bacterial community, suggesting an important role for protists in the fate of more-labile aquifer organic contaminants.

Helicobacter pylori adherence to gastric epithelial cells: a role for non-adhesin virulence genes

Helicobacter pylori is a major aetiological agent in gastroduodenal disorders and adherence of the bacteria to the gastric mucosa is one of the initial stages of infection. Although a number of specific adhesins has been identified, other H. pylori virulence factors may play a role in adherence to gastric epithelial cells directly or through interaction with other adhesins. This study assessed the effect of 16 H. pylori virulence factors on the adherence of the bacteria to gastric AGS cells and on gastric epithelial cell cycle distribution. Defined isogenic H. pylori SS1 mutants were used. After co-incubation of gastric AGS cells and bacteria, adherence of H. pylori to AGS cells was visualised by immunofluorescence microscopy and quantified by flow cytometry. Cell cycle phase distribution was analysed by flow cytometry with propidium iodide staining. Mutants were tested for their ability to adhere to AGS cells and compared with the wild-type SS1 strain. Mutations in genes in the cag pathogenicity island showed that cagP and cagE mutants adhered less than the wild-type strain to AGS cells, whereas a cagF mutant showed no reduction in adherence. Mutations in genes involved in flagellar biosynthesis showed that the adherence ability of fliQ, fliM and fliS mutants was reduced, but a flhB mutant possessed wild-type levels of adherence. Mutations in genes coding for the urease (ureB) and phospholipase (pldA) enzymes did not affect adherence, but mutation of the tlyA gene encoding an H. pylori haemolysin resulted in a reduced adherence. A fliQ mutant, with reduced adherence to AGS cells, was less able to induce AGS cell apoptosis than SS1. The ability to induce G0G1 cell cycle arrest was also abolished in the fliQ mutant. However, an increased cell number in S phase was observed when AGS cells were exposed to the fliQ mutant compared with SS1, suggesting that unattached bacteria may still be able to stimulate cell proliferation. In addition to known adhesins, other bacterial virulence factors such as CagE, CagP, FliQ, FliM, FliS and TlyA appear to play a role in H. pylori adherence to gastric epithelial cells. Mutations in these genes may affect H. pylori pathogenicity by reducing either the ability of the bacteria to attach to gastric epithelial cells or the intensity of bacteria-host cell interactions.