An in vitro biofilm model system to facilitate study of microbial communities of the human oral cavity.

The human oral cavity is host to a diverse microbiota. Much of what is known about the behaviour of oral microbes derives from studies of individual or several cultivated species, situations which do not totally reflect the function of organisms within more complex microbiota or multispecies biofilms. The number of validated models that allow examination of the role that biofilms play during oral cavity colonization is also limited. The CDC biofilm reactor is a standard method that has been deployed to study interactions between members of human microbiotas allowing studies to be completed during an extended period under conditions where nutrient availability, and washout of waste products are controlled. The objective of this work was to develop a robust in vitro biofilm-model system from a pooled saliva inoculum to study the development, reproducibility and stability of the oral microbiota. By employing deep sequencing of the variable regions of the 16S rRNA gene, we found that the CDC biofilm reactor could be used to efficiently cultivate microbiota containing all six major phyla previously identified as the core saliva microbiota. After an acclimatisation period, communities in each reactor stabilised. Replicate reactors were predominately populated by a shared core microbiota; variation between replicate reactors was primarily driven by shifts in abundance of shared operational taxonomic units. We conclude that the CDC biofilm reactor can be used to cultivate communities that replicate key features of the human oral cavity and is a useful tool to facilitate studies of the dynamics of these communities.

Antimicrobial Activity of the Quinoline Derivative HT61 against Staphylococcus aureus Biofilms.

Staphylococcus aureus biofilms are a significant problem in health care settings, partly due to the presence of a nondividing, antibiotic-tolerant subpopulation. Here we evaluated treatment of S. aureus UAMS-1 biofilms with HT61, a quinoline derivative shown to be effective against nondividing Staphylococcus spp. HT61 was effective at reducing biofilm viability and was associated with increased expression of cell wall stress and division proteins, confirming its potential as a treatment for S. aureus biofilm infections.

Antibiotic-loaded synthetic calcium sulfate beads for prevention of bacterial colonization and biofilm formation in periprosthetic infections.

Periprosthetic infection (PI) causes significant morbidity and mortality after fixation and joint arthroplasty and has been extensively linked to the formation of bacterial biofilms. Poly(methyl methacrylate) (PMMA), as a cement or as beads, is commonly used for antibiotic release to the site of infection but displays variable elution kinetics and also represents a potential nidus for infection, therefore requiring surgical removal once antibiotics have eluted. Absorbable cements have shown improved elution of a wider range of antibiotics and, crucially, complete biodegradation, but limited data exist as to their antimicrobial and antibiofilm efficacy. Synthetic calcium sulfate beads loaded with tobramycin, vancomycin, or vancomycin-tobramycin dual treatment (in a 1:0.24 [wt/wt] ratio) were assessed for their abilities to eradicate planktonic methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis relative to that of PMMA beads. The ability of the calcium sulfate beads to prevent biofilm formation over multiple days and to eradicate preformed biofilms was studied using a combination of viable cell counts, confocal microscopy, and scanning electron microscopy of the bead surface. Biofilm bacteria displayed a greater tolerance to the antibiotics than their planktonic counterparts. Antibiotic-loaded beads were able to kill planktonic cultures of 10(6) CFU/ml, prevent bacterial colonization, and significantly reduce biofilm formation over multiple days. However, established biofilms were harder to eradicate. These data further demonstrate the difficulty in clearing established biofilms; therefore, early preventive measures are key to reducing the risk of PI. Synthetic calcium sulfate loaded with antibiotics has the potential to reduce or eliminate biofilm formation on adjacent periprosthetic tissue and prosthesis material and, thus, to reduce the rates of periprosthetic infection.

Contact-dependent growth inhibition causes reversible metabolic downregulation in Escherichia coli.

Contact-dependent growth inhibition (CDI) is a mechanism identified in Escherichia coli by which bacteria expressing two-partner secretion proteins encoded by cdiA and cdiB bind to BamA in the outer membranes of target cells and inhibit their growth. A third gene in the cluster, cdiI, encodes a small protein that is necessary and sufficient to confer immunity to CDI, thereby preventing cells expressing the cdiBA genes from inhibiting their own growth. In this study, the cdiI gene was placed under araBAD promoter control to modulate levels of the immunity protein and thereby induce CDI by removal of arabinose. This CDI autoinhibition system was used for metabolic analyses of a single population of E. coli cells undergoing CDI. Contact-inhibited cells showed altered cell morphology, including the presence of filaments. Notably, CDI was reversible, as evidenced by resumption of cell growth and normal cellular morphology following induction of the CdiI immunity protein. Recovery of cells from CDI also required an energy source. Cells undergoing CDI showed a significant, reversible downregulation of metabolic parameters, including aerobic respiration, proton motive force (Deltap), and steady-state ATP levels. It is unclear whether the decrease in respiration and/or Deltap is directly involved in growth inhibition, but a role for ATP in the CDI mechanism was ruled out using an atp mutant. Consistent with the observed decrease in Deltap, the phage shock response was induced in cells undergoing CDI but not in recovering cells, based on analysis of levels of pspA mRNA.

Removal of Dental Biofilms with an Ultrasonically Activated Water Stream.

Acidogenic bacteria within dental plaque biofilms are the causative agents of caries. Consequently, maintenance of a healthy oral environment with efficient biofilm removal strategies is important to limit caries, as well as halt progression to gingivitis and periodontitis. Recently, a novel cleaning device has been described using an ultrasonically activated stream (UAS) to generate a cavitation cloud of bubbles in a freely flowing water stream that has demonstrated the capacity to be effective at biofilm removal. In this study, UAS was evaluated for its ability to remove biofilms of the cariogenic pathogen Streptococcus mutans UA159, as well as Actinomyces naeslundii ATCC 12104 and Streptococcus oralis ATCC 9811, grown on machine-etched glass slides to generate a reproducible complex surface and artificial teeth from a typodont training model. Biofilm removal was assessed both visually and microscopically using high-speed videography, confocal scanning laser microscopy (CSLM), and scanning electron microscopy (SEM). Analysis by CSLM demonstrated a statistically significant 99.9% removal of S. mutans biofilms exposed to the UAS for 10 s, relative to both untreated control biofilms and biofilms exposed to the water stream alone without ultrasonic activation (P < 0.05). The water stream alone showed no statistically significant difference in removal compared with the untreated control (P = 0.24). High-speed videography demonstrated a rapid rate (151 mm(2) in 1 s) of biofilm removal. The UAS was also highly effective at S. mutans, A. naeslundii, and S. oralis biofilm removal from machine-etched glass and S. mutans from typodont surfaces with complex topography. Consequently, UAS technology represents a potentially effective method for biofilm removal and improved oral hygiene.

Significance of high soil lead concentrations for childhood lead burdens.

The lead exposure of children and their mothers has been studied in two towns with mean soil lead contents of 900 and 400 ppm. No significant difference in blood or fecal lead contents was demonstrated between the two populations, but a small difference in hair lead content was shown. The blood lead content of children was greater than that of their mothers and was higher in the summer than in the spring samples. Children with pica for soil in the control area had increased lead content of blood and hair. Preliminary data for children and mothers from villages with mean soil lead contents of 500 ppm and 10,000 ppm are reported which show significant differences in blood and hair lead content within the normal range. The data suggest that soil lead content of 10,000 ppm may result in increased absorption of lead in children, but to a degree which is unlikely to be of biological significance.

Geochemistry and health in the United Kingdom.

Before the 1960s, comparisons between the distribution of trace elements in the environment and health in the United Kingdom were primarily confined to ad hoc studies in areas associated with particular agricultural disorders or with unusual human mortality or morbidity records. More recently, increasing interest in the importance of trace elements in crop and animal production and in the hazards of environmental pollution have created a need for more systematic geochemical data. Geochemical reconnaissance maps for England, Wales, Northern Ireland and parts of Scotland have demonstrated the extent of many known clinical trace element problems in agriculture and have also been valuable in delineating areas within which subclinical disorders may occur. Their application to studies on the composition of soils, food crops and surface waters in relation to public health has proved encouraging. Current knowledge and present investigations into environmental geochemistry and human health in the U.K. are reviewed, together with future research requirements.

Regional geochemical mapping.

One of the prime requirements for effective study of environmental geochemistry in relation to health is the production of multi-element atlases showing the distribution of the elements on the regional scale. The choice of method for compiling such atlases can vary according to a number of geological, environmental and other factors. The overriding consideration, however, is to assist (in conjunction with other relevant sources of information) in defining, quickly and cheaply, potential problem areas wherein to concentrate more detailed studies to ensure maximum return from the funds and scientific manpower available. Numerous sampling and analytical techniques have been employed. Each technique and approach has its own scope, limitation and problems of interpretation. Whatever method is chosen, the use of computer-based statistical data reduction, analysis and map compilation is mandatory. Although it was apparent more than 20 years ago that geochemical atlases would eventually become a national cartographic requirement, regional geochemical mapping is still in the experimental stage. This trend is now evident in activity in a number of countries. The methods being employed, however, are so diverse that there is an urgent need for international collaboration aimed at securing data that are as mutually compatible as possible, having regard to the conditions, needs and resources of the individual countries involved.

Metal removal by sulphate-reducing bacteria from natural and constructed wetlands.

The use of wetlands is a promising technology to treat acid mine drainage, yet there is little understanding of the fundamental biological processes involved. They are considered to centre on the complex anaerobic ecology within sediments and involve the removal of metals by sulphate-reducing bacteria (SRB). These bacteria generate hydrogen sulphide and cause precipitation of metals from solution as the insoluble metal sulphide. Sulphate-reducing bacteria have been isolated from natural and constructed wetlands receiving acid mine drainage. Sulphide production by isolates and removal of the metals iron, manganese and zinc were measured, as well as utilization of a range of carbon sources. Marked ecological differences between the wetlands were reflected in population composition of SRB enrichments, and these consortia displayed significant differences in sulphide generation and rates of metal removal from solution. Rates of metal removal did not correlate with sulphide generation in all cultures, suggesting the involvement of other biological mechanisms of metal removal. Differences in substrate utilization have highlighted the need for further investigation of carbon flow and potential carbon sources within constructed wetlands.

Postmortem diagnosis of Factor V Leiden from paraffin wax embedded tissue.

Activated protein C resistance resulting from Factor V Leiden is an important inherited thrombophilia disorder which is found in 3.5% of people in the UK. The genetic defect can be detected using the PCR and the diagnosis can be made postmortem from paraffin wax embedded tissue. The presence of Factor V Leiden should be sought in all cases of unexplained sudden death resulting from venous thromboembolism.

Influence of surfaces on sulphidogenic bacteria.

Sulphidogenic bacteria in oil reservoirs are of great economic importance in terms of souring, fouling and corrosion. Mixed cultures containing these bacteria were isolated from chalk formations in North Sea oil reservoirs. These were thermophilic cultures, growing optimally at 60°C. Oil formations are porous matrices, providing a very large surface area and ideal conditions for bacterial attachment, survival and growth. This study included assessments of sulphide production rates of thermophilic (t-)sulphidogen consortia with and without additional surfaces. The availability of a surface contributed significantly to the rate and extent of sulphide generation. Surfaces were offered in varying amounts to growing planktonic cultures: significantly more sulphide was produced from cultures in contact with a surface than from identical cultures in the absence of a surface. In another series of experiments, t-sulphidogens were added to chalk rock chips in the presence of nutrients and incubated for several months. This resulted in rapid sulphide generation, the final concentration being related to the initial nutrient concentration. Subsequent nutrient addition resulted in renewed sulphide generation. It is suggested that bacteria in reservoirs can withstand long periods of nutrient deprivation while attached within the porous rock matrix and opportunistically utilise nutrients when they become available.

Fungal colonization and biodeterioration of plasticized polyvinyl chloride.

Significant substratum damage can occur when plasticized PVC (pPVC) is colonized by microorganisms. We investigated microbial colonization of pPVC in an in situ, longitudinal study. Pieces of pPVC containing the plasticizers dioctyl phthalate and dioctyl adipate (DOA) were exposed to the atmosphere for up to 2 years. Fungal and bacterial populations were quantified, and colonizing fungi were identified by rRNA gene sequencing and morphological characteristics. Aureobasidium pullulans was the principal colonizing fungus, establishing itself on the pPVC between 25 and 40 weeks of exposure. A group of yeasts and yeast-like fungi, including Rhodotorula aurantiaca and Kluyveromyces spp., established themselves on the pPVC much later (after 80 weeks of exposure). Numerically, these organisms dominated A. pullulans after 95 weeks, with a mean viable count +/- standard error of 1,000 +/- 200 yeast CFU cm(-2), compared to 390 +/- 50 A. pullulans CFU cm(-2). No bacterial colonization was observed. We also used in vitro tests to characterize the deteriogenic properties of fungi isolated from the pPVC. All strains of A. pullulans tested could grow with the intact pPVC formulation as the sole source of carbon, degrade the plasticizer DOA, produce extracellular esterase, and cause weight loss of the substratum during growth in vitro. In contrast, several yeast isolates could not grow on pPVC or degrade DOA. These results suggest that microbial succession may occur during the colonization of pPVC and that A. pullulans is critical to the establishment of a microbial community on pPVC.

Plasticizers increase adhesion of the deteriogenic fungus Aureobasidium pullulans to polyvinyl chloride.

Initial adhesion of fungi to plasticized polyvinyl chloride (pPVC) may determine subsequent colonization and biodeterioration processes. The deteriogenic fungus Aureobasidium pullulans was used to investigate the physicochemical nature of adhesion to both unplasticized PVC (uPVC) and pPVC containing the plasticizers dioctyl phthalate (DOP) and dioctyl adipate (DOA). A quantitative adhesion assay using image analysis identified fundamental differences in the mechanism of adhesion of A. pullulans blastospores to these substrata. Adhesion to pPVC was greater than that to uPVC by a maximum of 280% after a 4-h incubation with 10(8) blastospores ml(-1). That plasticizers enhance adhesion to PVC was confirmed by incorporating a dispersion of both DOA and DOP into the blastospore suspension. Adhesion to uPVC was increased by up to 308% in the presence of the dispersed plasticizers. Hydrophobic interactions were found to dominate adhesion to uPVC because (i) a strong positive correlation was observed between substratum hydrophobicity (measured by using a dynamic contact angle analyzer) and adhesion to a range of unplasticized polymers including uPVC, and (ii) neither the pH nor the electrolyte concentration of the suspension buffer, both of which influence electrostatic interactions, affected adhesion to uPVC. In contrast, adhesion to pPVC is principally controlled by electrostatic interactions. Enhanced adhesion to pPVC occurred despite a relative reduction of 13 degrees in the water contact angle of pPVC compared to that of uPVC. Furthermore, adhesion to pPVC was strongly dependent on both the pH and electrolyte concentration of the suspension medium, reaching maximum levels at pH 8 and with an electrolyte concentration of 10 mM NaCl. Plasticization with DOP and DOA therefore increases adhesion of A. pullulans blastospores to pPVC through an interaction mediated by electrostatic forces.

Green fluorescent protein as a novel indicator of antimicrobial susceptibility in Aureobasidium pullulans.

Presently there is no method available that allows noninvasive and real-time monitoring of fungal susceptibility to antimicrobial compounds. The green fluorescent protein (GFP) of the jellyfish Aequoria victoria was tested as a potential reporter molecule for this purpose. Aureobasidium pullulans was transformed to express cytosolic GFP using the vector pTEFEGFP (A. J. Vanden Wymelenberg, D. Cullen, R. N. Spear, B. Schoenike, and J. H. Andrews, BioTechniques 23:686-690, 1997). The transformed strain Ap1 gfp showed bright fluorescence that was amenable to quantification using fluorescence spectrophotometry. Fluorescence levels in Ap1 gfp blastospore suspensions were directly proportional to the number of viable cells determined by CFU plate counts (r(2) > 0.99). The relationship between cell viability and GFP fluorescence was investigated by adding a range of concentrations of each of the biocides sodium hypochlorite and 2-n-octylisothiozolin-3-one (OIT) to suspensions of Ap1 gfp blastospores (pH 5 buffer). These biocides each caused a rapid (< 25-min) loss of fluorescence of greater than 90% when used at concentrations of 150 microg of available chlorine ml(-1) and 500 microg ml(-1), respectively. Further, loss of GFP fluorescence from A. pullulans cells was highly correlated with a decrease in the number of viable cells (r(2) > 0.92). Losses of GFP fluorescence and cell viability were highly dependent on external pH; maximum losses of fluorescence and viability occurred at pH 4, while reduction of GFP fluorescence was absent at pH 8.0 and was associated with a lower reduction in viability. When A. pullulans was attached to the surface of plasticized poly(vinylchloride) containing 500 ppm of OIT, fluorescence decreased more slowly than in cell suspensions, with > 95% loss of fluorescence after 27 h. This technique should have broad applications in testing the susceptibility of A. pullulans and other fungal species to antimicrobial compounds.