Fluorescence-based in situ assay to probe the viability and growth kinetics of surface-adhering and suspended recombinant bacteria (original) (raw)
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Applied and Environmental Microbiology, 2007
For quantification of bacterial adherence to biomaterial surfaces or to other surfaces prone to biofouling, there is a need for methods that allow a comparative analysis of small material specimens. A new method for quantification of surface-attached biotinylated bacteria was established by in situ detection with fluorescencelabeled avidin-D. This method was evaluated utilizing a silicon wafer model system to monitor the influences of surface wettability and roughness on bacterial adhesion. Furthermore, the effects of protein preadsorption from serum, saliva, human serum albumin, and fibronectin were investigated. Streptococcus gordonii, Streptococcus mitis, and Staphylococcus aureus were chosen as model organisms because of their differing adhesion properties and their clinical relevance. To verify the results obtained by this new technique, scanning electron microscopy and agar replica plating were employed. Oxidized and poly(ethylene glycol)-modified silicon wafers were found to be more resistant to bacterial adhesion than wafers coated with hydrocarbon and fluorocarbon moieties. Roughening of the chemically modified surfaces resulted in an overall increase in bacterial attachment. Preadsorption of proteins affected bacterial adherence but did not fully abolish the influence of the original surface chemistry. However, in certain instances, mostly with saliva or serum, masking of the underlying surface chemistry became evident. The new bacterial overlay method allowed a reliable quantification of surface-attached bacteria and could hence be employed for measuring bacterial adherence on material specimens in a variety of applications.
Real time noninvasive monitoring of contaminating bacteria in a soft tissue implant infection model
Journal of Biomedical Materials Research Part B: Applied Biomaterials, 2009
Infection is the main cause of biomaterials-related failure. A simple technique to test in-vivo new antimicrobial and/or nonadhesive implant coatings is unavailable. Current in vitro methods for studying bacterial adhesion and growth on biomaterial surfaces lack the influence of the host immune system. Most in vivo methods to study biomaterials-related infections routinely involve implant-removal, preventing comprehensive longitudinal monitoring. In vivo imaging circumvents these drawbacks and is based on the use of noninvasive optical imaging of bioluminescent bacteria. Staphylococcus aureus Xen29 is genetically modified to be stably bioluminescent, by the introduction of a modified full lux operon onto its chromosome. Surgical meshes with adhering S. aureus Xen29 were implanted in mice and bacterial growth and spread into the surrounding tissue was monitored longitudinally from bioluminescence with a highly sensitive CCD camera. Distinct spatiotemporal bioluminescence patterns, extending beyond the mesh area into surrounding tissues were observed. After 10 days, the number of living organisms isolated from explanted meshes was found to correlate with bioluminescence prior to sacrifice of the animals. Therefore, it is concluded that in vivo imaging using bioluminescent bacteria is ideally suited to study antimicrobial coatings taking into account the host immune system. In addition, longitudinal monitoring of infection in one animal will significantly reduce the number of experiments and animals. '
Surface enhanced bacterial fluorescence and enumeration of bacterial adhesion
Biofouling, 2013
The use of flow displacement systems for studying initial bacterial adhesion to surfaces is mostly confined to transparent substrata. The objective of this study is to present a method based on macroscopic fluorescence imaging to enumerate adhering fluorescent bacteria on non-transparent substrata, realtime and under flow. To this end, a stepwise protocol is described to quantitate adhesion of green-fluorescent-protein producing Staphylococcus aureus on polished and non-polished metal and polymer surfaces accounting for surface-enhanced-fluorescence on metal surfaces, quantitated by the ratio of the single cell fluorescence observed for adhering and planktonic bacteria. Enumeration of adhering fluorescent staphylococci by the proposed method coincides with results obtained using metallurgical microscopy. As an advantage however, non-homogeneous surface coverage and surface roughness do not limit the applicability of the method. Moreover, the accurate quantitation of surface-enhanced-fluorescence arising from adhering bacteria offers a new pathway to evaluate bacterial cell surface deformation during adhesion.
Nanoscience-Led Antimicrobial Surface Engineering to Prevent Infections
ACS Applied Nano Materials, 2021
One of the major complications associated with the implantation of biomedical devices regardless of their function is biomaterial associated infection. Infections are generally initiated by opportunistic bacterial colonization and biofilm development on the surface of implanted biomaterials, rendering the infection impervious to host defenses and antimicrobials. Moreover, the infection around soft tissues also has a significant role in biomaterial-associated infections. It is well documented that the nature of an implant infection is influenced by the design and composition of the implant biomaterial, host environment, clinical procedure and patient hygiene. Herein, we explore the adhesion mechanisms of bacteria to the biomaterials and review systematic antimicrobial strategies to reduce the contamination of biomaterials and underlying implant infection using Staphylococcus aureus as a model bacterial pathogen. Also, we discuss the preventive and therapeutic strategies and explain the future perspectives for the development of nanoscience-based strategies for the engineering of antimicrobial surfaces, including nanostructure surface, microbe-surface interactions, synthetic nanostructured surfaces, dynamic surfaces with antifouling agents, coated surfaces with antimicrobial properties (polymer coating, surface release active coating).
Quantitation of Bacterial Adhesion to Polymer Surfaces by Bioluminescence
Zentralblatt für Bakteriologie, 1998
Quantitation of microbes adhering to a surface is commonly used in studies of micro bial adhesion to different surfaces. We have quantified different staphylococcal strains adhering to polymer surfaces by measuring bacterial ATP (adenosine triphosphate) by bioluminescence. The method is sensitive, having a detection limit of 10 4 bacterial cells. Viable counting of bacterial cells may yield falsely low results due to the presence of "dormant" and adherent bacteria. By using bioluminescence, this can be avoided. Cells of different bacterial species and cells of strains of the same species were shown to dif fer significantly in their basal ATP content (8.7 X 10-13 -5.2 X 10-22 MATP). The size of adherent and planktonic bacteria decreased with time (0.7 !lm~ 0.3 !lm, 20 days). During incubation in nutrient-poor buffer ("starvation"), the ATP content of adherent bacteria decreased after 24-96 h whereas that of planktonic bacteria was stable over 20 days. The presence of human serum or plasma did not interfere significantly with the test results. Since the ATP concentration of bacterial strains of different species var ies and is also influenced by the growth conditions of bacteria (solid or liquid culture medium), a species-specific standard curve has to be established for bacteria grown under the same culture conditions. We conclude that the method is a sensitive tool to quantify adherent bacteria during experiments lasting for less than 6 h and constitutes a valuable method to be used in conjunction with different microscopical techniques.
Escherichia coli adhesion, biofilm development and antibiotic susceptibility on biomedical materials
Journal of biomedical materials research. Part A, 2015
The aim of this work was to test materials typically used in the construction of medical devices regarding their influence in the initial adhesion, biofilm development and antibiotic susceptibility of Escherichia coli biofilms. Adhesion and biofilm development was monitored in 12-well microtiter plates containing coupons of different biomedical materials-silicone (SIL), stainless steel (SS) and polyvinyl chloride (PVC)-and glass (GLA) as control. The susceptibility of biofilms to ciprofloxacin and ampicillin was assessed, and the antibiotic effect in cell morphology was observed by scanning electron microscopy. The surface hydrophobicity of the bacterial strain and materials was also evaluated from contact angle measurements. Surface hydrophobicity was related with initial E. coli adhesion and subsequent biofilm development. Hydrophobic materials, such as SIL, SS, and PVC, showed higher bacterial colonization than the hydrophilic GLA. Silicone was the surface with the greatest numbe...
Eur Cell Mater, 2004
This article reviews the mechanisms of bacterial adhesion to biomaterial surfaces, the factors affecting the adhesion, the techniques used in estimating bacteria-material interactions and the models that have been developed in order to predict adhesion. The process of bacterial adhesion includes an initial physicochemical interaction phase and a late molecular and cellular one. It is a complicated process influenced by many factors, including the bacterial properties, the material surface characteristics, the environmental factors, such as the presence of serum proteins and the associated flow conditions. Two categories of techniques used in estimating bacteria-material interactions are described: those that utilize fluid flowing against the adhered bacteria and counting the percentage of bacteria that detach, and those that manipulate single bacteria in various configurations which lend themselves to more specific force application and provide the basis for theoretical analysis of the receptor-ligand interactions. The theories that are reviewed are the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, the thermodynamic approach and the extended DLVO theory. Over the years, significant work has been done to investigate the process of bacterial adhesion to biomaterial surfaces, however a lot of questions still remain unanswered.