Journal of Molecular Biology
ReviewThe Limitations of In Vitro Experimentation in Understanding Biofilms and Chronic Infection
Graphical abstract
Introduction
Bacteria were once thought to exist as single, free-floating planktonic cells that are community independent. John William (Bill) Costerton changed this perception in the late 1970s when he observed surface-associated microbial aggregates enclosed within a matrix of extracellular material, a phenomena he later termed “biofilm” [1], [2]. Today, the biofilm phenotype has been identified in up to 80% of all non-acute infections, including foreign-body-related infections, otitis media, orthopaedic wounds, catheter infections, chronic wounds and lung-related infections [3], [4], [5], [6], [7]. The interchange between planktonic and biofilm phenotypes is believed, but not proven, to commonly manifest clinically as acute and chronic infections, respectively.
Acute infections tend to be fast spreading with a rapid onset. They are often controlled by the host immune response, and excessive intervention is not always required. However, if host defences fail and therapeutic intervention is required, acute infections can usually be cleared within days [8]. Conversely, chronic infections are where there is a delay in the healing process (an inability of the injured site to restore anatomical and functional integrity), consistent with the severity of the injury [9]. The presence of biofilms and their innate ability to tolerate antibiotics up to 1000 times greater than planktonic cells is thought to delay wound restoration [10], [11], [12], [13]. Cells assuming the biofilm phenotype are commonly observed in patients with various underlying conditions, which can be system wide in the case of immunodeficiency and diabetes or can be more focused in the case of venous leg ulcers and cystic fibrosis (CF). In the case of CF, chronic biofilm infections have been known to persist in the airways for over 30 years. Therefore, chronic infections are an ever-increasing problem due to their recalcitrance towards extensive antibiotic treatment regimes and persistence under sustained attack from the host's innate and adaptive immune response systems [10], [14].
The treatment and management of patients suffering from chronic infections represents a significant monetary and labour-intensive burden to healthcare providers. Recently, the direct costs of chronic infections, such as those affecting the dermis, were estimated to be in excess of $18 billion, affecting 2 million residents in the United States alone and resulting in 200,000 deaths annually [15]. These data relate to one country and a single infected organ. If this is representative of other countries and other conditions, then chronic infections represent a huge worldwide problem. A recent report on antimicrobial resistance (AMR) has stated that we can expect up to 10 million extra deaths annually worldwide by 2050 due to AMR [16]. The problem of chronic infection is only going to aid the rise of AMR.
Much of our current knowledge about chronic infection comes from studying bacteria growing in test tubes. Costerton encouraged laboratories worldwide to deviate from studying planktonic cultures and instead focus on understanding surface-associated biofilms. This has become increasingly more relevant as we battle with the issues posed by AMR. We are becoming increasingly aware of significant differences that exist between in vitro biofilms grown in the laboratory and in vivo biofilms found during actual infection. This raises the question as to whether the experiments that we currently perform in the laboratory are useful for understanding how bacterial biofilms form and contribute to AMR during infection.
To understand the biology of infection better, we need another paradigm shift, a new wave of methods and experiments that better represent clinical conditions (of which biofilm formation is only one aspect). The use of some methods can potentially hamper our understanding of various aspects of infection, as they do not always accurately represent what we observe clinically. This review summarises what we know about bacteria during infection and how our current in vitro methods fail to represent such factors. In the following sections, we discuss biofilms and polymicrobial interactions, particularly in the context of Pseudomonas aeruginosa as one of the most common opportunistic pathogens that cause chronic infection. We explore the differences between in vitro and in vivo observations, and we discuss how to better bridge the gap between the two, increasing experimental accuracy so that our benchside data can be used to improve bedside treatment.
Section snippets
The Role of Biofilms during Chronic Infection
Chronic infections persist despite apparently adequate antibiotic therapy and in the face of the host's innate and adaptive defence mechanisms. Chronic infections are characterised by persistent and progressive pathology, mainly due to the inflammatory response surrounding in vivo biofilms [17]. This biofilm lifestyle appears to impair the host's ability to combat the infectious agent. The innate immune response in the form of recruitment of neutrophils and their inability to break through the
In Vitro Investigation of Biofilms
During the last three decades, biofilms of pathogenic species have been extensively studied by a wide range of research groups, each with differing objectives, but all with the same overall aim—to expand our knowledge on biofilms to better understand infection. Using continuous flow cell conditions and confocal laser scanning microscopy (CLSM), we are closer to understanding the processes involved in the initial attachment of cells to surfaces in vitro [46], [47], [48]. Combining molecular
In Vivo Investigation of Biofilms
More recently, the advent of in vivo methods have increased our understanding of biofilms during chronic infection. There are a range of in vivo models that simulate chronic infections, such as surface wounds [75], [76], subcutaneous wounds [77], [78], implant-related wounds (such as catheter, orthopaedic and dental) [79], [80], [81], [82], [83], otitis media [84], [85], [86] and CF [87], [88], [89], [90] to name but a few. As with all models, some are deemed more applicable than others. For
In Vivo Conditions and In Vitro Methods
Sometimes it may not be ethical, practicable or feasible to conduct in vivo experimentation. Given the issues highlighted previously, how can we better represent in vivo conditions in our in vitro models? It is widely known, for P. aeruginosa at least, that different nutritional cues result in altered biofilm formation, virulence, motility and QS [46], [113], [114], [115], [116], [117]. These differences become increasingly important when factors of clinical relevance, such as virulence and
Conclusion and Recommendations
Most of our mechanistic knowledge and hypotheses surrounding biofilm formation and how this relates to chronic infection is based upon in vitro observations, primarily through the use of microtitre plate assays and flow cell systems. These systems have greatly enhanced our knowledge about the mechanisms of how cells attach to surfaces and differentiate into multicellular biofilms. However, it is becoming increasingly apparent that many of our in vitro methods do not accurately represent in vivo
Acknowledgements
This work was funded by a Human Frontier Science grant to S.P.D. and T.B. (RGY0081/2012).
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