The process of surface adhesion and biofilm development is a survival strategy employed by virtually all bacteria and refined over millions of years. This process is designed to anchor microorganisms in a nutritionally advantageous environment and to permit their escape to greener pastures when essential growth factors have been exhausted. Bacterial attachment to a surface can be divided into several distinct phases, including primary and reversible adhesion, secondary and irreversible adhesion, and biofilm formation. Each of these phases is ultimately controlled by the expression of one or more gene products. Ultrastructurally, the mature bacterial biofilm resembles an underwater coral reef containing pyramidal or mushroom-shaped microcolonies of organisms embedded within an extracellular glycocalyx, with channels and cavities to allow the exchange of nutrients and waste. The biofilm protects its inhabitants from predators, dehydration, biocides, and other environmental extremes while regulating population growth and diversity through primitive cell signals. From a physiological standpoint, surface-bound bacteria behave quite differently from their planktonic counterparts. Recognizing that bacteria naturally occur as surface-bound and often polymicrobic communities, the practice of performing antimicrobial susceptibility tests using pure cultures and in a planktonic growth mode should be questioned. That this model does not reflect conditions found in nature might help explain the difficulties encountered in the management and treatment of biomedical implant infections.

/pdf/bacterial-adhesion-seen-any-good-biofilms-lately-2f7wm9gh5j.pdf

Bacterial Adhesion: Seen Any Good Biofilms Lately?

Candida albicans is implicated in many biomaterial-related infections Typically, these infections are associated with biofilm formation Cells in biofilms display phenotypic traits that are dramatically different from those of their free-floating planktonic counterparts and are notoriously resistant to antimicrobial agents Consequently, biofilm-related infections are inherently difficult to treat and to fully eradicate with normal treatment regimens Here, we report a rapid and highly reproducible microtiter-based colorimetric assay for the susceptibility testing of fungal biofilms, based on the measurement of metabolic activities of the sessile cells by using a formazan salt reduction assay The assay was used for in vitro antifungal susceptibility testing of several C albicans strains grown as biofilms against amphotericin B and fluconazole and the increased resistance of C albicans biofilms against these antifungal agents was demonstrated Because of its simplicity, compatibility with a widely available 96-well microplate platform, high throughput, and automation potential, we believe this assay represents a promising tool for the standardization of in vitro antifungal susceptibility testing of fungal biofilms

Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms.

Observing naturally occurring biofilms in situ or ex situ has revealed the wide distribution of sessile microbial communities. The ubiquity, variety and complexity of biofilms is now widely accepted by microbiologists. While they are associated with many beneficial functions such as nutrient cycling, bioremediation and colonization resistance, adverse effects including recalcitrance, their involvement in industrial fouling, contamination and infection have made biofilms a priority research topic. We know that most biofilms, other than within certain infections and laboratory flasks, are composed of multiple species and that there is arguably no unifying biofilm architecture. Biofilms do however share certain properties including the presence of gradients of nutrients, gasses and metabolic products, relatively increased cell density, deposition of extracellular polymeric substances and marked recalcitrance towards antimicrobial treatments. Much of our understanding of biofilm physiology and micro-ecology originates from experiments using in vitro biofilm models. Broadly speaking, such models may be used to replicate environmental conditions within the laboratory or to focus on selected variables such a growth rate or fluid flow, etc. This chapter provides an overview of some commonly used biofilm models including microtitre plate systems, flow cells, the constant depth film fermenter, annular reactors and the perfused biofilm fermenter. While perfused biofilm fermenters, in particular, enable growth rate to be controlled within thin, relatively homogenous, quasi steady-state biofilms through modulation of flow rate nutrient availability, other models provide representative modelling of in situ conditions where steady states may be uncommon.

Chapter 4 In Vitro Biofilm Models. An Overview

The chromosomal multiple antibiotic resistance operon, mar, is widely represented amongst Gram-negative bacteria and has been implicated in resistance towards oxidative stress agents, organic solvents and a large number of structurally unrelated antimicrobial agents. The major mechanism associated with such increased resistance is an upregulation of the efflux pump acrAB. Growth as a biofilm is often associated with similar generalized reductions in susceptibility to inimical agents. Escherichia coli K12 (AG100), an isogenic mutant of AG100 constitutive for mar expression (AG102) and an isolate deleted of the mar locus (MCH164) were grown as biofilms in cellulose-fibre depth filters and perfused with a simple salts, minimal medium (CDM) over 120 h. Biofilms were exposed to various concentrations of ciprofloxacin (0.004, 0.015 and 0.1 mg/L) for 42 h. The numbers of viable cells within the perfusate and within the biofilm were estimated throughout. Whereas no differences were seen between the wild-type and mar-deleted isolates, that constitutive for mar displayed reduced susceptibility to ciprofloxacin at concentrations of 0.004 mg/L (MIC for AG100 was 0.0052 mg/L). Similar antibiotic perfusion experiments were conducted using isolates in which the efflux pump acrAB was either deleted (AG100-A) or constitutively expressed (AG100-B). Exposure of AG100-A biofilms to ciprofloxacin at 0.004 and 0.1 mg/L showed similar susceptibilities to those seen in the wildtype (AG100) and mar-deleted (MCH164) isolates and suggested that acrAB was not induced within the attached population. On the other hand, constitutive expression of acrAB (AG100-B) protected biofilms against the lower concentration of ciprofloxacin used (0.004 mg/L). This protection was again lost at concentrations of 0.1 mg/L. Overall, these results show that ciprofloxacin resistance in biofilms is not mediated by the upregulation of the mar or acrAB operons.

An evaluation of the potential of the multiple antibiotic resistance operon (mar) and the multidrug efflux pump acrAB to moderate resistance towards ciprofloxacin in Escherichia coli biofilms

Bioelectrochemical systems (BES) represent a wide range of different biofilm-based bioreactors that includes microbial fuel cells (MFCs), microbial electrolysis cells (MECs) and microbial desalination cells (MDCs). The first described bioelectrical bioreactor is the Microbial Fuel Cell and with the exception of MDCs, it is the only type of BES that actually produces harvestable amounts of electricity, rather than requiring an electrical input to function. For these reasons, this review article, with previously unpublished supporting data, focusses primarily on MFCs. Of relevance is the architecture of these bioreactors, the type of membrane they employ (if any) for separating the chambers along with the size, as well as the geometry and material composition of the electrodes which support biofilms. Finally, the structure, properties and growth rate of the microbial biofilms colonising anodic electrodes, are of critical importance for rendering these devices, functional living ‘engines’ for a wide range of applications.

Microbial fuel cells and their electrified biofilms.

A method for the study of membrane-associated biofilm populations of Escherichia coli ATCC 8739 using modified Swinnex filter units was developed. Biofilms were established under carbon limitation in a chemically defined simple salts medium. Cells, pressure filtered on to cellulose nitrate membranes in situ, were perfused from the sterile side. Steady-state conditions were attained at which the growth rate of the sessile cells could be demonstrated to be proportional to the flow rate of medium. The antibiotic susceptibility of these biofilms was examined by including ciprofloxacin within the perfusing medium. Susceptibility of the biofilms to ciprofloxacin was found to be affected not only by its concentration, but also by the growth rate of the bacteria.

/pdf/the-development-of-a-small-scale-biofilm-model-suitable-for-216djacyd5.pdf

The development of a small-scale biofilm model suitable for studying the effects of antibiotics on biofilms of gram-negative bacteria.

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The development of a small-scale biofilm model suitable for studying the effects of antibiotics on biofilms of gram-negative bacteria.