Review
In vitro and in vivo model systems to study microbial biofilm formation

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Abstract

Biofilm formation is often considered the underlying reason why treatment with an antimicrobial agent fails and as an estimated 65–80% of all human infections is thought to be biofilm-related, this presents a serious challenge. Biofilm model systems are essential to gain a better understanding of the mechanisms involved in biofilm formation and resistance. In this review a comprehensive overview of various in vitro and in vivo systems is presented, and their advantages and disadvantages are discussed.

Introduction

Since 1943, when marine microbiologist Claude ZoBell described the so-called “bottle effect” (referring to the phenomenon that the number of free-living microorganisms in fresh sea water gradually declines when the water is kept in a glass bottle, while the number of attached microorganisms increases) (ZoBell, 1943) we have been aware of the fact that microorganisms are capable of living their life attached to a surface. However, it then took more than 30 years (and the paradigm-changing work of Bill Costerton and colleagues) to accept that for microorganisms (both bacteria and fungi) the biofilm mode of life is the rule rather than the exception (Costerton et al., 1978, Costerton et al., 1999). Biofilms are defined as consortia of microorganisms that are attached to a biotic or abiotic surface. Biofilm formation is a multi-stage process in which microbial cells adhere to the surface (initial reversible attachment), while the subsequent production of an extracellular matrix (containing polysaccharides, proteins and DNA) results in a firmer attachment (Sauer, 2003, Stoodley et al., 2002). Cells embedded in this matrix communicate with each other and show a coordinated group behaviour mediated by a process called quorum sensing (QS) (Zhang and Dong, 2004). Sessile (biofilm-associated) cells are phenotypically and physiologically different from non-adhered (planktonic) cells and one of the typical properties of sessile cells is their increased resistance to antimicrobial agents (Donlan and Costerton, 2002, Mah and O'Toole, 2001, Stewart and Costerton, 2001). Biofilm formation is often considered the underlying reason why treatment with an antimicrobial agent fails and as an estimated 65–80% of all infections is thought to be biofilm-related, this presents a serious challenge (Costerton et al., 1999, Hall-Stoodley et al., 2004, Parsek and Singh, 2003). Biofilm formation can also have detrimental effects in industrial systems. Biofouling is especially problematic in systems in which materials come into contact with water, including heat exchangers, ship hulls and (marine) fish cages (Braithwaite and McEvoy, 2005, Coetser and Cloete, 2005, Flemming, 2002). Of particular relevance to human health is biofilm formation in drinking water reservoirs and distribution systems as these biofilms hinder the efficient operation of these systems. In addition, they may also pose a health risk to the users, providing a habitat for pathogenic miroorganisms like Legionella pneumophila and Escherichia coli (Flemming, 2002, Juhna et al., 2007). On the other hand, there are many (potential) applications of microbial biofilms, in processes as diverse as bioremediation (Singh et al., 2006), production of fine chemicals (Li et al., 2006), fermentation (Kunduru and Pometto, 1996), biofiltration (Cohen, 2001), wastewater treatment (Nicolella et al., 2000), biofuel production (Wang and Chen, 2009) and generation of electricity in microbial fuel cells (Rabaey et al., 2007).

In order to increase our knowledge concerning biofilm biology, biofilm model systems to be used for the study of the often complex communities under controlled conditions are indispensable (Doyle, 1999, Hamilton et al., 2003, Wolfaardt et al., 2007). In this review we present an overview of in vitro and in vivo model systems and discuss their advantages and disadvantages. The focus of this review is on tools to study medically-relevant biofilms, but many of the models can of course also be used to mimick biofilm formation in other settings.

Section snippets

Microtiter plate-based model systems

Microtiter plate (MTP)-based systems are among the most-frequently-used biofilm model systems (see for example Cerca et al., 2005, Christensen et al., 1985, Coenye and Peeters Nelis, 2007, De Prijck et al., 2007, Gabrielson et al., 2002, Krom et al., 2007, Miyake et al., 1992, Peeters et al., 2008a, Peeters et al., 2008b, Peeters et al., 2008c, Pettit et al., 2005, Pitts et al., 2003, Ramage et al., 2001, Shakeri et al., 2007, Stepanovic et al., 2000, Toté et al., 2008, Silva et al., 2010,

Caenorhabditis elegans model

Studies using the C. elegans model system usually focus on virulence as such (i.e. determining whether infection results in reduced survival of the worms) and/or on the effect of particular chemical compounds on this survival. However, a number of studies employing C. elegans have specifically dealt with microbial biofilm formation. The first indication that bacteria can form biofilms in C. elegans came from the study by Darby et al. (2002) with Yersinia pestis. In this study it was shown that

In vitro models

Recent evidence has shown that also in chronic wounds (e.g. diabetic foot ulcers, pressure ulcers) microbial biofilms (often polymicrobial) can be found. It has been hypothesised that these biofilms and the lack of their elimination by leukocytes are responsible for the chronic nature of the infection (Bjarnsholt et al., 2008, James et al., 2008). The first chronic wound biofilm model was developed at the Medical Biofilm Research Institute in Lubbock (Texas, USA) and was aptly named the Lubbock

Quantification and visualisation of biofilms grown in various model systems

Following biofilm growth in an in vitro or in vivo model system, the extent of biofilm formation can be measured in a variety of ways. Before providing a brief overview of the different approaches available, we want to stress the importance of standardising the techniques used to recover biofilm-grown cells from the surface. For example, it has been shown that the passage of a liquid–air interface (e.g. an air bubble) can result in considerable detachment and often-used procedures like dipping

Which model system to choose?

The goal of this review was to present an overview of commonly used in vitro and in vivo biofilm model systems, their potential applications, and their advantages and disadvantages.

It is obvious that MTP-based systems permit a higher throughput, are generally less labour-intensive, do not require specialised equipment and are cheaper. These systems allow “multiplexing” (i.e. multiple organisms and/or treatments can be included in a single run) and as such are very well-suited for screening

Acknowledgements

We wish to thank BOF-UGent and the Fund for Scientific Research-Flanders for financial support and Drs. Kristof De Prijck, Heleen Nailis and Elke Peeters for valuable contributions.

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