High-throughput metal susceptibility testing of microbial biofilms

BACKGROUND: Microbial biofilms exist all over the natural world, a distribution that is paralleled by metal cations and oxyanions. Despite this reality, very few studies have examined how biofilms withstand exposure to these toxic compounds. This article describes a batch culture technique for biofilm and planktonic cell metal susceptibility testing using the MBEC assay. This device is compatible with standard 96-well microtiter plate technology. As part of this method, a two part, metal specific neutralization protocol is summarized. This procedure minimizes residual biological toxicity arising from the carry-over of metals from challenge to recovery media. Neutralization consists of treating cultures with a chemical compound known to react with or to chelate the metal. Treated cultures are plated onto rich agar to allow metal complexes to diffuse into the recovery medium while bacteria remain on top to recover. Two difficulties associated with metal susceptibility testing were the focus of two applications of this technique. First, assays were calibrated to allow comparisons of the susceptibility of different organisms to metals. Second, the effects of exposure time and growth medium composition on the susceptibility of E. coli JM109 biofilms to metals were investigated. RESULTS: This high-throughput method generated 96-statistically equivalent biofilms in a single device and thus allowed for comparative and combinatorial experiments of media, microbial strains, exposure times and metals. By adjusting growth conditions, it was possible to examine biofilms of different microorganisms that had similar cell densities. In one example, Pseudomonas aeruginosa ATCC 27853 was up to 80 times more resistant to heavy metalloid oxyanions than Escherichia coli TG1. Further, biofilms were up to 133 times more tolerant to tellurite (TeO(3)(2-)) than corresponding planktonic cultures. Regardless of the growth medium, the tolerance of biofilm and planktonic cell E. coli JM109 to metals was time-dependent. CONCLUSION: This method results in accurate, easily reproducible comparisons between the susceptibility of planktonic cells and biofilms to metals. Further, it was possible to make direct comparisons of the ability of different microbial strains to withstand metal toxicity. The data presented here also indicate that exposure time is an important variable in metal susceptibility testing of bacteria

Virulence gene expression by staphylococcus epidermidis biofilm cells exposed to antibiotics

Staphylococcus epidermidis have become important causes of nosocomial infections, as its pathogenesis is correlated with the ability to form biofilms on polymeric surfaces. Production of poly-N-acetylglucosamine (PNAG) is crucial for S. epidermidis biofilm formation and is synthesized by the gene products of the icaADBC gene cluster. Production of PNAG/polysaccharide intercellular adhesin and biofilm formation are regulated by the alternative sigma factor, σB, and is influenced by a variety of environmental conditions including disinfectants and other antimicrobial substances. The susceptibility of five S. epidermidis strains to antibiotics alone and in double combination was previously tested. Our results demonstrated that some combinations are active and present a general broad spectrum against S. epidermidis biofilms, namely rifampicin–clindamycin and rifampicin–gentamicin. In the present study, it was investigated whether the combination of rifampicin with clindamycin and gentamicin and these antibiotics alone influence the expression of specific genes (icaA and rsbU) of S. epidermidis within biofilms using real-time polymerase chain reaction. The data showed that in most cases the expression of both genes tested significantly increased after exposure to antimicrobial agents alone and in combination. Besides having a similar antimicrobial effect, rifampicin combined with clindamycin and gentamicin induced a lower expression of biofilm-related genes relatively to rifampicin alone. Associated with the advantage of combinatorial therapy in avoiding the emergence of antibiotic resistance, this study demonstrated that it can also cause a lower genetic expression of icaA and rsbU genes, which are responsible for PNAG/polysaccharide intercellular adhesin production, and consequently reduce biofilm formation recidivism, relatively to rifampicin alone.F. Gomes and P. Teixeira fully acknowledge the financial support of Fundacao para a Ciencia e Tecnologia through the grants SFRH/BD/32126/2006 and SFRH/BPD/26803/2006, respectively

Effect of single versus antibiotic combinations on Staphylococcus epidermidis biofilm viability and on genetic expression of some virulence genes

In this study five clinical isolates strains were used, and nine antibiotics at breakpoint concentrations: vancomycin, tetracycline, rifampicin, gentamicin, cefazolin, cephalothin, levofloxacine, daptomycin and clindamycin were tested. 48 hours biofilms were grown on Calgary Biofilm Device (CBD) and challenged overnight with antibiotics alone and in combination. Biofilm cells viability was determined by colony forming units (cfu). Afterwards, the effect of the most active antibiotics combinations against S. epidermidis biofilm on genetic expression of some genes of interest such as: icaA, icaR, sarA and rsbU was determined by real-time PCR. Although biofilms were generally insensitive to individual antibiotics, they were more susceptible to combinations. Levofloxacine was a constituent of almost all the combinations active against S. epidermidis biofilm pointing to be part of any antibiotic therapy directed against biofilms of these organisms

Polymer-supported metal catalysts for the heterogeneous polymerisation of lactones

A series of metal complexes were immobilised onto an inert poly(styrene) (PS) support and utilised in the solvent free ring-opening polymerisation (ROP) of various lactones. PS-LHZnOAc, PS-LHSnOct and PS-LClSnOct were identified as the most successful heterogeneous catalysts for the ROP of L-lactide. Investigations by in situ ATR-FT-IR revealed conversions reaching ca. 90% in 6, 2.3 hours and 55 minutes, respectively, with excellent molecular weight control and dispersities (ĐM 1.15–1.17). Catalyst loadings as low as 15 ppm metal and TOF values of up to 810 h−1 could also be achieved. Higher molecular weights could be targeted (ca. 35 kDa) whilst maintaining low dispersities in comparison to the industrial standard. Catalyst reuse was also possible, with up to 7 reuse cycles, albeit accompanied by a progressive reduction in conversion. Energy-Dispersive X-ray (EDX) spectroscopy and Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES) showed low metal content in the unpurified polymer (as low as 335 ppm, similar to what is found in polymer purified by classical methods), suggesting these systems as promising reusable catalysts for the industrial production of metal-free renewable polymers

The use of microscopy and three-dimensional visualization to evaluate the structure of microbial biofilms cultivated in the Calgary Biofilm Device

Microbes frequently live within multicellular, solid surface-attached assemblages termed biofilms. These microbial communities have architectural features that contribute to population heterogeneity and consequently to emergent cell functions. Therefore, three-dimensional (3D) features of biofilm structure are important for understanding the physiology and ecology of these microbial systems. This paper details several protocols for scanning electron microscopy and confocal laser scanning microscopy (CLSM) of biofilms grown on polystyrene pegs in the Calgary Biofilm Device (CBD). Furthermore, a procedure is described for image processing of CLSM data stacks using amira(™), a virtual reality tool, to create surface and/or volume rendered 3D visualizations of biofilm microorganisms. The combination of microscopy with microbial cultivation in the CBD – an apparatus that was designed for high-throughput susceptibility testing – allows for structure-function analysis of biofilms under multivariate growth and exposure conditions

Optical coherence tomography in the assessment of acute changes in cutaneous vascular diameter induced by heat stress.

There are limited imaging technologies available that can accurately assess or provide surrogate markers of the in vivo cutaneous microvessel network in humans. In this study, we establish the use of optical coherence tomography (OCT) as a novel imaging technique to assess acute changes in cutaneous microvessel area density and diameter in humans. OCT speckle decorrelation images of the skin on the ventral side of the forearm up to a depth of 500 μm were obtained prior to and following 20-25 mins of lower limb heating in eight healthy males (30.3±7.6 yrs). Skin red blood cell flux was also collected using laser Doppler flowmetry probes immediately adjacent to the OCT skin sites, along with skin temperature. OCT speckle decorrelation images were obtained at both baseline and heating time points. Forearm skin flux increased significantly (0.20±0.15 to 1.75±0.38 CVC, P<0.01), along with forearm skin temperature (32.0±1.2 to 34.3±1.0°C, P<0.01). Quantitative differences in the automated calculation of vascular area densities (26±9 to 49±19%, P<0.01) and individual microvessel diameters (68±17 to 105±25 μm, P<0.01) were evident following the heating session. This is the first in vivo within-subject assessment of acute changes in the cutaneous microvasculature in response to heating in humans and highlights the use of OCT as an exciting new imaging approach for skin physiology and clinical research

Distinct Effects of Blood Flow and Temperature on Cutaneous Microvascular Adaptation

Aims: We performed two experiments to determine whether cutaneous microvascular adaptations in response to repeated core temperature elevation are mediated by increases in skin temperature, and/or, skin blood flow. Methods: Healthy subjects participated for 8-weeks in thrice-weekly bouts of 30mins lower limb heating (40°C). In Study 1, both forearms were “clamped” at basal skin temperature throughout each heating bout (n=9). Study 2 involved identical lower limb heating, with the forearms under ambient conditions (unclamped, n=10). In both studies, a cuff was inflated around one forearm during the heating bouts to assess the contribution of skin blood flow and temperature responses. We assessed forearm skin blood flow responses to both lower limb (systemic reflex) heating, and to local heating of the forearm skin, pre and post intervention. Results: Acutely, lower limb heating increased core temperature (Study 1: +0.63±0.15°C, Study 2: +0.69±0.19°C, P<0.001) and forearm skin blood flow (Study 1: 10±3 vs 125±44, Study 2: 16±9 vs 136±41 PU, P<0.001), with skin responses significantly attenuated in the cuffed forearm (P<0.01). Skin blood flow responses to local heating decreased in Study 1 (clamped forearms, week 0vs8: 1.46±0.52 vs 0.99±0.44 CVC, P<0.05), whereas increases occurred in Study 2 (unclamped; week 0vs8: 1.89±0.57 vs 2.27±0.52 CVC, P<0.05). Cuff placement abolished local adaptations in both studies. Conclusion: Our results indicate that repeated increases in skin blood flow and skin temperature result in increased skin flux responses to local heating, whereas repeated increases in skin blood flow in the absence of change in skin temperature induced the opposite response. Repeated increases in core temperature induce intrinsic microvascular changes, the nature of which are dependent upon both skin blood flow and skin temperature