A Glance at Biofilm Study Approaches

Despite their ubiquity and ability to colonize every biotope on the planet, in vitro replication of complex biofilm and infections is a challenging task. Scientists set up different protocols and methods to simulate these complex structures in the laboratory, but none is fully predictive or relevant for comprehensive analysis [i].

Biofilms are characterized by a complex and heterogeneous structure, strongly influenced by endogenous and exogenous factors. Because of their adaptive strategies, structural complexity and organization across different kinds of strains, we can compare biofilms with an animal organ, with its functions, tissues, and specific characteristics. The ability to adhere to surfaces essentially depends on the species of bacteria, the composition and nature of the surface, nutrient and water availability, motility of free cells, and cell-to-cell communication. The molecular interactions between cells seems to be regulated by quorum sensing, a chemical communication system that allows regulation of gene expression in a cell density-dependent manner. The switch from planktonic to sessile communities is finely regulated from a genetic point of view. The second messenger c-di-GMP is a key player, modulating this formation process. Motility and virulence are also important factors [ii,iii]. Mature biofilms typically consist of differentiated structures of several kinds of microbial cells embedded in their matrix or slime [iv,v], composed by an aqueous mixture of polysaccharides, proteins, nucleic acids, and other substances essential for the biofilm growth and survival. This matrix is responsible for structural integrity and organization of biofilms and is different among different kind of microbial communities [iv, vi]. The three-dimensional structure of EPS could be very complex, and in some cases, it is possible to distinguish water-filled channels involved in elaborated metabolic pathways. In certain conditions during detachment from biofilms, microbes can release free cells into the environment to colonize virgin areas and start new biofilm formation cycles [vii]. Moreover, encased in their niche, microbial cells can exchange genetic material contributing to insurgence of resistance.

Single-species biofilms are laboratory artifacts, used to simplify research in this field. Instead, microorganisms live in complicate and structured consortia, where intricate spatiotemporal dynamics are driven by interactions among different species. The spatial arrangement of biofilms can determine survival of different species and is a form of adaptation to the surrounding conditions. In natural environments, a multispecies microbial community can be represented as a city of bacteria that stay and live together, sharing their genetic material [viii]. This association allows for metabolic cooperation and increased resistance to antibiotics or host immunity in the case of pathogenic biofilms [ix]. These organized communities ensure an ecological equilibrium, and they can survive under stressful conditions such as low water and nutrient availability [x]. This high level of structural complexity needs specialization and sophisticated systems of cell-cell signals [xi]. The mechanisms involved in this complex community formation are not fully understood. For scientists, the challenge is to reproduce lab-scale biofilm models that are representative of real-life conditions to fully understand their physiology [xii] and find effective therapies to eradicate highly structured and pathogenic biofilms.

Another characteristic that makes biofilm so harmful and difficult to eradicate is the presence of persister cells. First identified by J. Bigger in 1944, these are inner cells, less metabolically active than the outer cells [xiii] and able to survive in the presence of bactericidal antibiotics and harsh conditions, such as oxidative stress, lack of nutrients, and attack from the host immune system. Persister cells may play a major role in recalcitrance mechanisms. Vulin et al (2018) provided evidence that, in clinical infections, they can manifest as small-colony variants (SCV) under stress conditions [xiv,xv,xvi].

Results of antibiofilm tests are strongly dependent on the platform used for the assay [xvii,xviii,xix], making reproducibility a challenge [xx]. To gain more solid and homogenous data on the effectiveness of an agent, different, complementary experiments need to be performed, as each approach evaluates on a specific biofilm feature [xxi]. This is also true for the detection methods used (see Figure). All systems and models have advantages and limitations with regard to the time needed to perform the experiment, the presence of unculturable strains [xxii], the need to distinguish dead and alive cells or different strains in multispecies biofilms [xxiii], the use of laboratory-attenuated strains versus clinically isolated strains [xiv], etc. Based on the needs and methods of each research project, some devices are more suitable than others [xv]. Several authors [i,xvi] have methodically reviewed the most used biofilm growth devices and the related detection tools. Laboratories often need to integrate these techniques, comparing intra and inter-laboratories results. Overall, a standardized protocol would be advisable if researchers want to compare outcomes coming from different sources [xvii,xviii].

A recent improvement introduced to prevent failure of in vivo assays is the study of microcosms, models that mimic in situ conditions, in addition to ex vivo models [xxix]. Indeed, in real-life infections, animal or host immune and inflammatory responses and patient health state influence both therapeutic activity and biofilm complexity [xxx,xxxi]. Moreover, biofilms show different characteristics in vivo than in vitro, such as size and shape [xvii,xxxii,xxxiii]. However, high cost, intrinsic complexity, invasiveness, and variability of in vivo models heavily limit their wide-scale use [xxv,xxxiv]. As a consequence, reproducibility from in vitro to in vivo settings is difficult and several antibiofilm strategies end up failing when tested in vivo [xxxiv,xxxv]. In addition, not all approaches that were successful in animal models show the same results in clinical trials [vii,xxxii].

The intrinsic complexity of biofilm systems partly explains the gap between clinical testing and basic research and the related challenge in obtaining data from basic research that are predictive of clinical test results.

A multitude of innovative antibiofilm strategies can be found in basic research papers. Only a small fraction (3.0%) of these, though, eventually reach the market and are translated into the clinical practice. Many approaches that are successful in experiments at the bench don’t make it past that stage and clinical scale-up remains limited. This suggests that innovative therapies (such as the use of phages or NPs) need more robust preclinical testing to assess their biocompatibility and effectiveness [xxxvi,xxxvii]. Recently, some innovative solutions have been moving towards preclinical assessment, as some small companies are taking on high-risk strategies and the related translational challenges [xxxviii]