Biofilm-associated infections are difficult to treat and eradicate because of their increased antimicrobial tolerance. In vitro biofilm models have enabled the high throughput testing of an array of differing novel antimicrobials and treatment strategies. However, biofilms formed in these oftentimes basic in vitro systems do not resemble biofilms seen in vivo. As a result, translatability from the lab to the clinic is poor or limited. To improve translatability, in vitro models must better recapitulate the host environment. This review describes and critically evaluates new and innovative in vitro models that better mimic the environments of a variety of clinically important, biofilm-associated infections of the skin, oropharynx, lungs, and infections related to indwelling implants and medical devices. This review highlights that many of these models represent considerable advances in the field of biofilm research and help to translate laboratory findings into the clinical practice.

Plasma-activated water (PAW) is an emerging sanitizer in the fresh food industry, where food safety must be achieved without a thermal kill step. Here, we demonstrate PAW generated in situ to be a competitive sanitizer technology, providing a significant reduction of pathogenic and spoilage microorganisms while maintaining the quality and shelf life of the produce item.

Healing and treatment of chronic wounds are often complicated due to biofilm formation by pathogens. Here, the efficacy of plasma activated water (PAW) as a pre-treatment strategy has been investigated prior to the application of topical antiseptics polyhexamethylene biguanide, povidone iodine, and MediHoney, which are routinely used to treat chronic wounds. The efficacy of this treatment strategy was determined against biofilms of Escherichia coli formed on a plastic substratum and on a human keratinocyte monolayer substratum used as an in vitro biofilm-skin epithelial cell model. PAW pre-treatment greatly increased the killing efficacy of all the three antiseptics to eradicate the E. coli biofilms formed on the plastic and keratinocyte substrates. However, the efficacy of the combined PAW-antiseptic treatment and single treatments using PAW or antiseptic alone was lower for biofilms formed in the in vitro biofilm-skin epithelial cell model compared to the plastic substratum. Scavenging assays demonstrated that reactive species present within the PAW were largely responsible for its anti-biofilm activity. PAW treatment resulted in significant intracellular reactive oxygen and nitrogen species accumulation within the E. coli biofilms, while also rapidly acting on the microbial membrane leading to outer membrane permeabilisation and depolarisation. Together, these factors contribute to significant cell death, potentiating the antibacterial effect of the assessed antiseptics.

Background: It has been proposed that GAS may form biofilms. Biofilms are microbial communities that aggregate on a surface, and exist within a self-produced matrix of extracellular polymeric substances. Biofilms offer bacteria an increased survival advantage, in which bacteria persist, and resist host immunity and antimicrobial treatment. The biofilm phenotype has long been recognized as a virulence mechanism for many Gram-positive and Gram-negative bacteria, however very little is known about the role of biofilms in GAS pathogenesis.Objective: This review provides an overview of the current knowledge of biofilms in GAS pathogenesis. This review assesses the evidence of GAS biofilm formation, the role of GAS virulence factors in GAS biofilm formation, modelling GAS biofilms, and discusses the polymicrobial nature of biofilms in the oropharynx in relation to GAS.Conclusion: Further study is needed to improve the current understanding of GAS as both a monospecies biofilm, and as a member of a polymicrobial biofilm. Improved modelling of GAS biofilm formation in settings closely mimicking in vivo conditions will ensure that biofilms generated in the lab closely reflect those occurring during clinical infection.

Biofilm formation on surfaces, tools and equipment can damage their quality and lead to high repair or replacement costs. Plasma-activated water (PAW), a new technology, has shown promise in killing biofilm and non-biofilm bacteria due to reactive oxygen and nitrogen species (RONS), particularly superoxide. However, the exact genetic mechanisms behind PAW’s effectiveness against biofilms remain unclear. Here, we examined the stress responses of Escherichia coli biofilms exposed to sub-lethal PAW treatment using bulk RNA sequencing and transcriptomics. We compared gene expression in PAW-treated E. coli biofilms with and without superoxide removal, achieved by adding the scavenger Tiron. Biofilms treated with PAW exhibited a 40 % variation in gene expression compared to those treated with PAW-Tiron and controls. Specifically, PAW treatment resulted in 478 upregulated genes (>1.5 log₂FC) and 186 downregulated genes (<−1.5 log₂FC) compared to the control. Pathway and biological process enrichment analysis revealed significant upregulation of genes involved in sulfur metabolism, ATP-binding transporter, amino acid metabolism, hypochlorite response systems and oxidative phosphorylation in PAW-treated biofilms compared to control. Biofilm viability and intracellular RONS accumulation were tested for E. coli mutants lacking key genes from these pathways. Knockout mutants of thioredoxin (trxC), thiosulfate-binding proteins (cysP), and NADH dehydrogenase subunit (nuoM) showed significantly reduced biofilm viability after PAW treatment. Notably, ΔtrxC biofilms had the highest intracellular ROS accumulation, as revealed by 2′,7′–dichlorofluorescin diacetate staining after PAW treatment. This confirms the importance of these genes in managing oxidative stress caused by PAW and highlights the significance of superoxide in PAW's bactericidal effects. Overall, our findings shed light on the specific genes and pathways that help E. coli biofilms survive and respond to PAW treatment, offering a new understanding of plasma technology and its anti-biofilm mechanisms.