How to study biofilms: technological advancements in clinical biofilm research

Abstract

Biofilm formation is an important survival strategy commonly used by bacteria and fungi, which are embedded in a protective extracellular matrix of organic polymers. They are ubiquitous in nature, including humans and other animals, and they can be surface- and non-surface-associated, making them capable of growing in and on many different parts of the body. Biofilms are also complex, forming polymicrobial communities that are difficult to eradicate due to their unique growth dynamics, and clinical infections associated with biofilms are a huge burden in the healthcare setting, as they are often difficult to diagnose and to treat. Our understanding of biofilm formation and development is a fast-paced and important research focus. This review aims to describe the advancements in clinical biofilm research, including both in vitro and in vivo biofilm models, imaging techniques and techniques to analyse the biological functions of the biofilm.

Keywords: biofilm; biofilm analysis; biofilm imaging; biofilm model; host-microbe interactions; infection.

Figure 1

Different methods of biofilm growth. Biofilms can be grown in a number of different ways. Biofilms can be grown statically in (A) microtitre plates and (B) microtitre plates with glass beads, titanium discs or hydroxyapatite discs in the bottom of the well. They can be grown dynamically in (C) flow cells combined wuth peristaltic pumps, (D) in a constant depth film fermenter, and (E) in a microfluidic system such as the BioFlux. The inoculum can range from (F) single and (G) mixed species polymicrobial consortia, and inoculating collected samples from participants, such as (H) collected saliva. In vivo modelling is generally peformed on (I) live animals, and ex vivo modelling is performed on extracted samples such as (J) teeth and (K) skin samples, and also skin models. Created with BioRender.com.

Figure 2

Images of biofilms using different techniques. (A) CLSM image of a 10-day old microcosm biofilm grown on a hydroxyapatite disc from a saliva sample inoculum. Biofilm is stained with LIVE/DEAD™ BacLight™ Bacterial Viability Kit (green - live; red - dead). (B) CLSM image of a mature 4-day old E. coli W strain biofilm grown under shear flow with a BioFlux microfluidic system. Bacterial membranes are stained with FilmTracer™ FM™ 1-43 dye. (C) Negative stain SEM image of a 1-day old enterotixogenic E. coli (ETEC) H10407 strain biofilm showing microcolony formation. (D) Cryo-EM derived atomic structure of bundled in situ archaeal bundling pili from Pyrobaculum calidifontis at 4 Å resolution (PDB ID: 7ueg) (Wang et al., 2022) . One extended pilus (purple) is surrounded by five other pili running in the opposite direction (green).

Figure 3

Spatial and functional analysis of biofilms. Imaging can be coupled with transcriptomics and mass spectrometry to assess the functionality of biofilm samples. (A) A schematic view of RAINBOW-seq, where different fluorescent dyes are sequentially added to a biofilm which is imaged at regular intervals, after which the biofilm is subjected to FACS cell sorting and then low quantity mRNA sequencing is performed which is mapped back to the image using bioinformatic gating parameters (Dar et al., 2021) . (B) A schematic view of imaging mass spectroscopy, where agar grown colony biofilms that are eGFP fluorescence tagged are grown and cryosectioned into thin slices, which are imaged and then subjected to MALDI-ToF mass spectrometry with post-processing mapping to the image. Created with BioRender.com.