Bacteria have evolved remarkable ways to survive and thrive in hostile environments. One of their most impressive strategies is forming biofilms—dense, multi-layered communities where bacteria live together, clinging to surfaces, and producing a protective matrix. These biofilms allow bacteria to resist antibiotics and evade the immune system, making them a significant challenge in treating infections.

A new tool called BaSSSh-seq (Bacterial Single-Cell RNA Sequencing) developed at the University of Nebraska Medical Center provides groundbreaking insights into how individual bacterial cells behave within these biofilms. Researchers applied this method to study Staphylococcus aureus, a notorious pathogen that forms biofilms on medical devices and in chronic wounds. Here’s what they uncovered:

BaSSSh-seq enables bacterial scRNA-seq of biofilm and incorporates rRNA depletion

A Split-pool barcoding attaches a combination of three barcodes to intracellular RNA transcripts of fixed and permeabilized cells. The 5′ end of the terminal barcode oligo also includes a UMI, PCR handle, and biotin tag. B Following lysis, streptavidin magnetic beads are used to purify captured transcripts. Then double-stranded cDNA is synthesized via random primer second strand synthesis and PCR amplification. C Substantial rRNA depletion is performed using an enzyme-free dual-strand subtractive hybridization technique, where biotin-tagged oligos specific to 5S, 16S, and 23S rRNA fragments are annealed to each cDNA strand and magnetically removed with streptavidin beads. The rRNA content can be lowered from >90% to <50% (****, p-value < 0.0001 by unpaired t-test). Data includes 4 biological replicates with no depletion (25,000 cells per sample library, 1.5 × 10⁵–2.5 × 10⁵ paired-end reads per sample) and 6 biological replicates with depletion (120,000–150,000 cells per sample library, 2.4 × 10⁷–4.4 × 10⁷ paired-end reads per sample). Data are presented as mean ± standard deviation. D Libraries are constructed for Illumina sequencing through fragmentation, ligation, and amplification to generate constructs containing P5/P7 ends with unique i5/i7 index combinations. 

Why Biofilms Are So Tough

Biofilms are not uniform. Instead, they are composed of subpopulations of bacteria that behave differently from one another. This diversity makes it difficult to treat biofilm-related infections because some bacteria may enter dormant states, resist antibiotics, or adapt to immune attacks.

Using BaSSSh-seq, the researchers were able to explore the genetic activity of single bacterial cells within a biofilm. They found that compared to free-floating (planktonic) bacteria, those within a biofilm display far greater variability in how their genes are turned on or off. This heterogeneity is key to the biofilm’s ability to adapt and survive under stress.

Mapping the Path from Free-Floating to Biofilm Growth

The researchers identified genetic pathways that guide bacteria as they transition from a planktonic state to becoming part of a biofilm. This “trajectory” includes turning on genes related to stress responses, metabolism, and biofilm formation, while silencing genes that promote free-floating growth. Visualizing these changes helps us understand how bacteria commit to the biofilm lifestyle and resist external threats.

Immune Cells and Biofilm Responses

Another fascinating discovery was how biofilms respond to immune system attacks. Different types of immune cells, such as macrophages and neutrophils, triggered specific changes in the biofilm’s gene activity. For instance, bacteria within biofilms ramped up stress responses and altered their metabolism when exposed to these immune cells. These adaptations likely help the biofilm survive immune pressure, offering clues into why biofilm infections are so persistent.

What This Means for Future Treatments

By revealing the inner workings of biofilms at the single-cell level, BaSSSh-seq opens up new opportunities for combating these tough bacterial communities. Researchers can now pinpoint which genes and pathways are critical for biofilm survival and target them with novel antibiotics or therapeutic strategies. Additionally, understanding how biofilms respond to immune attacks could inspire new ways to boost the body’s defenses against infections.

Final Thoughts

The ability to study bacteria at single-cell resolution is a game-changer in microbiology. Tools like BaSSSh-seq allow us to explore the hidden complexities of biofilms, shedding light on their resilience and adaptability. With further research, this technology could lead to innovative treatments that dismantle biofilms and improve outcomes for patients suffering from chronic infections.