Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide, causing invasive diseases such as pneumonia, sepsis and meningitis, but also less severe localised infections like otitis media. To enable pneumococcal survival in the nutritionally challenging nasopharyngeal niche, it possesses an extensive repertoire of genes dedicated to the utilisation of a wide range of carbohydrates. Galactose, the predominant carbohydrate found in the respiratory tract, is important in pneumococcal colonisation and virulence, and therefore represents an attractive target for the development of new treatments. In this study we investigated the potential of fluorinated galactose analogues as a strategy for preventing and treating pneumococcal biofilms. The analogues were tested against planktonic pneumococci, and their effect on in vitro biofilms assessed through biomass and viability measurements with validation through confocal and scanning electron microscopy. Their effect on adhesion of pneumococcus to Detroit562 pharyngeal epithelial cells and cytotoxicity was also assessed. As anticipated, galactose fluorinated at the C-3 (Gal-3F) and C-6 (Gal-6F) positions had no effect on planktonic growth within glucose-rich BHI medium, as pneumococcus utilises glucose as the primary carbohydrate during this mode of growth. Correlating with the transition towards biofilm formation involving a switch to galactose metabolism, 1-2% w/v Gal-3F successfully inhibited biofilm formation for four out of five strains tested; however, no activity was observed against 48-hour established biofilms and there was no inhibition of epithelial adhesion. The equivalent concentrations of Gal-6F had no effect on pneumococcal biofilms, suggesting that the site of fluorination is important in activity. The inhibition of biofilm formation by Gal-3F confirms that targeting galactose metabolism is a potentially viable strategy for treating pneumococcal infection, and that the rational design of fluorinated glycomimetics could offer an alternative approach to targeting pneumococcal biofilms.

Perfluorocarbon nanodroplets (PFC-NDs) are promising ultrasound-responsive theranostic agents with applications in both diagnostic imaging and drug delivery. The acoustic vaporisation threshold, extravasation potential, and stability of PFC-NDs are all affected by their size. However, methods to ensure reproducible size and concentration during production by sonication are lacking. To address this need, we examined the effect of temperature, sonication time, sonication intensity, PFC concentration and sonicator tip height on ND characteristics. PFC-NDs with a perfluoro-n-pentane (PFP) core and a phospholipid shell were manufactured by probe-sonication. Pulsed sonication was used to maintain the sample temperature below the boiling point of PFP. Median particle diameter was measured using nanoparticle tracking analysis. PFC-ND diameter increased with increasing PFP concentration, with a stronger relationship as sonicator tip height increased. Above 5% v/v PFP, there was a qualitative increase in the number of particles visible by light microscopy. Increasing the sonication duration did not yield a significant change in ND size. A minimum amplitude of 60% was required for mixing to occur, with amplitudes of 80% and 100% resulting in foam production. Sonicator power output was linear with respect to time but differed depending on sample volume, composition, and vessel geometry. This study indicates that controlling the processing parameters can facilitate reproducible manufacturing of PFC-NDs.

Sulphate reducing prokaryotes are widely acknowledged as key contributors to microbiologically influenced corrosion in industry. Characterisation of their behaviour within mixed-species biofilms that reflect ecologically relevant conditions is limited. A novel dual anaerobic biofilm reactor protocol allowed a complex microbial consortium to be investigated. Continual biofilm growth resulted in significantly greater corrosive pit density, with 15 and 47 pits mm-2 in the biotic reactor for as received (AR) and polished (P) coupons respectively. There was an average pit density of 3 pits mm-2 in the abiotic reactor for both AR and P coupons. Moreover, a greater pit depth and size were observed when compared to the sterile abiotic conditions. Identifying and understanding the relative contributions of different microbial mechanisms within mixed-species biofilms is critical. Importantly, electroactive and corrosive Desulfovibrio desulfuricans and Desulfovibrio vulgaris were identified within the biofilm. These microorganisms play a crucial role in extracellular electron transfer, a key process in microbiologically influenced corrosion. The protocol not only deepens the mechanistic understanding of MIC but also offers a versatile tool for testing mitigation strategies under realistic and customizable conditions. This integrated approach can ultimately support the development of more targeted, sustainable corrosion prevention and management practices.

ABSTRACT Oilfield systems are a multifaceted ecological niche, which consistently experiences microbiologically influenced corrosion. However, simulating the environmental conditions of an offshore system within the laboratory is notoriously difficult. A novel dual anaerobic biofilm reactor protocol allowed a complex mixed-species marine biofilm to be studied. Interestingly, electroactive corrosive bacteria and fermentative electroactive bacteria growth was supported within the biofilm microenvironment. Critically, the biotic condition exhibited pits with a greater average area, which is characteristic of microbiologically influenced corrosion. This research seeks to bridge the gap between experimental and real-world scenarios, ultimately enhancing the reliability of biofilm management strategies in the industry. IMPORTANCE It is becoming more widely understood that any investigation of microbiologically influenced corrosion requires a multidisciplinary focus on multiple lines of evidence. Although there are numerous standards available to guide specific types of testing, there are none that focus on integrating biofilm testing. By developing a novel dual anaerobic reactor model to study biofilms, insights into the different abiotic and biotic corrosion mechanisms under relevant environmental conditions can be gained. Using multiple lines of evidence to gain a holistic understanding, more sustainable prevention and mitigation strategies can be designed. To our knowledge, this is the first time all these metrics have been combined in one unified approach. The overall aim of this paper was to explore recent advances in biofilm testing and corrosion research and provide recommendations for future standards being drafted. However, it is important to note that this article itself is not intending to serve as a standard.

Standard antibiotic susceptibility tests (ASTs) often fail to accurately predict treatment outcomes because they do not account for biofilm-specific mechanisms of reduced susceptibility. In the present study, we explored alternative approaches to predict tobramycin susceptibility of Pseudomonas aeruginosa biofilms that were experimentally evolved in physiologically relevant conditions. To this end, we used four analytical methods – whole-genome sequencing (WGS), matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), isothermal microcalorimetry (IMC) and multi-excitation Raman spectroscopy (MX-Raman). Machine learning models were trained on data outputs from these methods to predict tobramycin susceptibility of our evolved strains and subsequently validated with a collection of clinical isolates. For minimal inhibitory concentration (MIC) predictions of the evolved strains, the highest accuracy ±1 was achieved with MALDI-TOF MS (97.83 %), while for biofilm prevention concentration (BPC) predictions, Raman spectroscopy performed best with an accuracy ±1 of 80.43 %. Overall, all analytical methods demonstrated comparable predictive performance, showing their potential for improving biofilm AST.

Antibiotic susceptibility tests (ASTs) often fail to predict treatment outcomes because they do not account for biofilm-specific tolerance mechanisms. In the present study, we explored alternative approaches to predict tobramycin susceptibility of Pseudomonas aeruginosa biofilms that were experimentally evolved in physiologically relevant conditions. To this end, we used four analytical methods - whole-genome sequencing (WGS), matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), isothermal microcalorimetry (IMC) and multi-excitation Raman spectroscopy (MX-Raman). Machine learning models were trained on data outputs from these methods to predict tobramycin susceptibility of our evolved strains and validated with a collection of clinical isolates. For minimal inhibitory concentration (MIC) predictions of the evolved strains, the highest accuracy was achieved with MALDI-TOF MS (97.83%), while for biofilm prevention concentration (BPC) predictions, Raman spectroscopy performed best with an accuracy of 80.43%. Overall, all analytical methods demonstrated comparable predictive performance, showing their potential for improving biofilm AST.

Antimicrobial resistance (AMR) poses a global healthcare challenge, where overprescription of antibiotics contributes to its prevalence. We have developed a rapid multi-excitation Raman spectroscopy methodology (MX-Raman) that outperforms conventional Raman spectroscopy and enhances specificity. A support vector machine (SVM) model was used to identify 20 clinical isolates of Pseudomonas aeruginosa with an accuracy of 93% using MX-Raman. Antibiotic sensitivity profiles for tobramycin, ceftazidime, ciprofloxacin, and imipenem were generated for the bacterial strains and compared with their Raman spectral signatures using MX-Raman. The 20 clinical strains were distinguished according to AMR profiles. Nine models were assessed for AMR classification performance, and SVM performed best, classifying AMR profiles of each strain with 91-96% accuracy. These data provide the basis for a new rapid clinical diagnostic platform that could screen for bacterial infection and recommend effective antibiotic treatment ahead of confirmation by conventional techniques, improving clinical outcomes and reducing the spread of AMR.

Food safety relies on rapid detection methods and rigorous sampling of the food processing environment and is challenged by recurrent biofilm contamination and by sublethally injured bacteria that can evade detection. Bactiscan is investigated as an alternative detection approach, a macro-scale and reagentless device that detects microbial contamination through activating the green fluorescence of glycoproteins in the bacterial cell wall. The detection capability of Bactiscan was tested on foodborne pathogens Escherichia coli, Listeria monocytogenes, Salmonella enterica, and Staphylococcus aureus. Detection by Bactiscan was assessed using 3 independent observers viewing bacterial samples dried on stainless steel, using 3 biological repeats and 5 technical repeats. Detection by Bactiscan was possible to 1.20 * 106 colony forming units (CFU), compared to 1.36 * 104 CFU by ATP swab testing, where Bactiscan detection limits were defined by the concentration at which 50% of the samples were observed under illumination of the device. Heat-killed and chlorine-stressed E. coli and S. enterica caused a 2-log reduction in detection by ATP swab tests (p ≤ 0.05), while detection by Bactiscan was unaffected (p ≥ 0.05). Pathogen biofilms were detectable via Bactiscan with >80% accuracy at 4 days of growth; E. coli and L. monocytogenes biofilms were visible at 2 days of growth. In situ contamination studies determined that Bactiscan can detect microbial contamination on chicken, salmon, and yoghurt samples with stronger fluorescence than a competitor's UV torch. The presence of one of the pathogens on the food samples was confirmed by metagenome sequencing, determining that S. aureus was present in 7 samples out of 9 with a relative abundance of >0.5%. These data demonstrate that Bactiscan can effectively detect bacteria present in the food processing environment and can complement existing technologies to improve food industry cleaning practices and infection prevention.

Understanding biocide performance in mixed-species biofilms is critical to mitigating microbiologically influenced corrosion (MIC). In this study, a novel dual anaerobic biofilm reactor was used to evaluate glutaraldehyde efficacy under environmentally relevant conditions, using a complex microbial consortium from marine sediment. Despite biocide dosing, biofilms persisted and induced localized corrosion, indicating incomplete mitigation. Each biocide application led to an electronegative shift in E corr and a reduction in H 2 S concentration, suggesting partial suppression of microbial activity. Raman spectroscopy and profilometry revealed differences in corrosion product composition and pit morphology between biotic and abiotic systems. 16S rRNA sequencing showed enrichment of stress-tolerant genera, including Exiguobacterium and Serpentinicella, consistent with increased chemical tolerance. These findings highlight the limitations of conventional biocide strategies and demonstrate the need for adaptive, community-informed treatment approaches. The dual-reactor model provides a robust platform for future MIC standardization efforts and mechanistic investigation of biofilm resilience under anoxic conditions.

Continual challenges due to microbial corrosion are faced by the maritime, offshore renewable and energy sectors. Understanding the biofilm and microbiologically influenced corrosion interaction is hindered by the lack of robust and reproducible physical models that reflect operating environments. A novel dual anaerobic biofilm reactor, using a complex microbial consortium sampled from marine littoral sediment, allowed the electrochemical performance of UNS G10180 carbon steel to be studied simultaneously in anaerobic abiotic and biotic artificial seawater. Critically, DNA extraction and 16S rRNA amplicon sequencing demonstrated the principal biofilm activity was due to electroactive bacteria, specifically sulfate-reducing and iron-reducing bacteria.

Microbial biofilms are complex communities of microorganisms that exist in various environments, including natural and human-built systems and have a significant economic impact on a global scale. In addition to their detrimental impacts, biofilms have been extensively studied for their potential benefits. In agriculture, biofilms are considered key organisational structures of microbes, exploited as biofertilisers, biostimulants, and biocontrol agents, with the potential to enhance soil health and plant growth. Despite ongoing research, there are still large knowledge gaps in the understanding of the mechanisms governing interaction between biofilms and plants, and how these can be manipulated to promote sustainable agriculture. The UK’s National Biofilms Innovation Centre [NBIC] and Argentine researchers have established a dialogue aimed at addressing these gaps and improving agricultural productivity through the integration of new technologies that can promote soil health whilst reducing environmental impact. Future research collaborations between the two countries in this area could have significant benefits for global agricultural innovation and the development of sustainable food systems. This publication takes on a ‘white paper’ format, consolidating complex discussions from a workshop between NBIC and Argentine researchers. It offers a comprehensive summary encompassing the insights, perspectives, and outcomes generated during the discussions among the participants, pinpointing three key priority areas for collaborative activities that were identified: (1) Using plant root biofilm composition as a sensor for soil health and to optimise interventions, (2) Biofilms and soil health resilience in a changing environment, (3) Intelligent seeds and innovative / automated large-scale monitoring systems. For the three identified priority areas, the early engagement of end-users [farmers] will be paramount to maximise technology adoption. Commitment from the governments and support from funding bodies in both countries will be essential for the establishment of robust research programmes and long-term successful collaborations between researchers, industry and end users.

The marine cyanobacterium Prochlorococcus is a main contributor to global photosynthesis, whilst being limited by iron availability. Cyanobacterial genomes typically encode two different types of FutA iron binding proteins: periplasmic FutA2 ABC transporter subunits bind Fe(III), while cytosolic FutA1 binds Fe(II). Owing to their small size and their economized genome Prochlorococcus ecotypes typically possess a single futA gene. How the encoded FutA protein might bind different Fe oxidation states was previously unknown. Here we use structural biology techniques at room temperature to probe the dynamic behavior of FutA. Neutron diffraction confirmed four negatively charged tyrosinates, that together with a neutral water molecule coordinate iron in trigonal bipyramidal geometry. Positioning of the positively charged Arg103 side chain in the second coordination shell yields an overall charge-neutral Fe(III) binding state in structures determined by neutron diffraction and serial femtosecond crystallography. Conventional rotation X-ray crystallography using a home source revealed X-ray induced photoreduction of the iron center with observation of the Fe(II) binding state; here, an additional positioning of the Arg203 side chain in the second coordination shell maintained an overall charge neutral Fe(II) binding site. Dose series using serial synchrotron crystallography and an XFEL X-ray pump-probe approach capture the transition between Fe(III) and Fe(II) states, revealing how Arg203 operates as a switch to accommodate the different iron oxidation states. This switching ability of the Prochlorococcus FutA protein may reflect ecological adaptation by genome streamlining and loss of specialized FutA proteins. Significance Statement Oceanic primary production by marine cyanobacteria is a main contributor to carbon and nitrogen fixation. Prochlorococcus is the most abundant photosynthetic organism on Earth, with an annual carbon fixation comparable to the net global primary production from agriculture. Its remarkable ecological success is based on the ability to thrive in low nutrient waters. To manage iron limitation, Prochlorococcus possesses the FutA protein for iron uptake and homeostasis. We reveal a molecular switch in the FutA protein that allows it to accommodate binding of iron in either the Fe(III) or Fe(II) state using structural biology techniques at room temperature and provide a plausible mechanism for iron binding promiscuity.

Priority question exercises are increasingly used to frame and set future research, innovation and development agendas. They can provide an important bridge between the discoveries, data and outputs generated by researchers, and the information required by policy makers and funders. Microbial biofilms present huge scientific, societal and economic opportunities and challenges. In order to identify key priorities that will help to advance the field, here we review questions from a pool submitted by the international biofilm research community and from practitioners working across industry, the environment and medicine. To avoid bias we used computational approaches to group questions and manage a voting and selection process. The outcome of the exercise is a set of 78 unique questions, categorized in six themes: (i) Biofilm control, disruption, prevention, management, treatment (13 questions); (ii) Resistance, persistence, tolerance, role of aggregation, immune interaction, relevance to infection (10 questions); (iii) Model systems, standards, regulatory, policy education, interdisciplinary approaches (15 questions); (iv) Polymicrobial, interactions, ecology, microbiome, phage (13 questions); (v) Clinical focus, chronic infection, detection, diagnostics (13 questions); and (vi) Matrix, lipids, capsule, metabolism, development, physiology, ecology, evolution environment, microbiome, community engineering (14 questions). The questions presented are intended to highlight opportunities, stimulate discussion and provide focus for researchers, funders and policy makers, informing future research, innovation and development strategy for biofilms and microbial communities.

The switch between planktonic and biofilm lifestyle correlates with intracellular concentration of the second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP). While bacteria possess cyclase and phosphodiesterase enzymes to catalyse formation or hydrolysis of c-di-GMP, both enzymatic domains often occur in a single protein. It is tacitly assumed that one of the two enzymatic activities is dominant, and that additional domains and protein interactions enable responses to environmental conditions and control activity. Here we report the structure of the phosphodiesterase domain of the membrane protein RbdA (regulator of biofilm dispersal) in a dimeric, activated state and show that phosphodiesterase activity is controlled by the linked cyclase. The phosphodiesterase region around helices α5/α6 forms the dimer interface, providing a rationale for activation, as this region was seen in contact with the cyclase domain in an auto-inhibited structure previously described. Kinetic analysis supports this model, as the activity of the phosphodiesterase alone is lower when linked to the cyclase. Analysis of a computed model of the RbdA periplasmatic domain reveals an all-helical architecture with a large binding pocket that could accommodate putative ligands. Unravelling the regulatory circuits in multi-domain phosphodiesterases like RbdA is important to develop strategies to manipulate or disperse bacterial biofilms.

Biofilms are widely recognised as a contributing factor in significant problems currently facing human health and industry. The following paper summarises a round table forum held at the 2021 International Biodeterioration and Biodegradation Symposium which discussed the potential role of standards in biofilm research and industry innovation. Standards and other forms of best-practice guidance are reviewed in an academic research context as well as in relation to industry impacts and product development. The understanding of fundamental aspects of biofilms is rapidly evolving, driven in part by new analytical methods. However, the complex and multidisciplinary nature of biofilm-associated problems and typically limited training available for industry personnel tackling the associated issues often reduces the ability to provide best-practice solutions. As such it is argued that more effort needs to be made by both academia and industry experts to provide consensus and associated documentation on standard test methods or guidance documents related to studying and combating biofilms.

Pseudomonas aeruginosa is an opportunistic pathogen of high medical importance and is one of the main pathogens responsible for the mortality of patients with cystic fibrosis. In addition to inherited antibiotic resistance, P. aeruginosa can form biofilms, defined as communities of microorganisms embedded in a self-produced matrix of extracellular polymeric substances adhering to each other and/or to a surface.

The rapid emergence of bacterial resistance to antibiotics in current use is occurring worldwide and poses a significant threat to global healthcare systems. Recent research to identify new effective anti-bacterial agents has focused on regulatory pathways as targets for interference. Regulatory mechanisms employing intracellular Bis-(3′,5′) cyclic di-guanylate (c-di-GMP) as a secondary messenger represent a distinct category of subjects. This molecule, c-di-GMP, is present in nearly all bacterial species and plays a pivotal role in governing various biological processes, encompassing antibiotic resistance, biofilm formation, and virulence. Alteration of the cellular concentrations of the nucleotide through modulation of associated signaling pathways has the potential to reduce biofilm formation or increase susceptibility of the biofilm bacteria to antibiotics. Here, we have developed a screen for compounds that alter c-di-GMP levels in Pseudomonas aeruginosa in co-culture with bronchial epithelial cells. Through the assay of 200 natural compounds, we were able to identify several substances showing promising effects on P. aeruginosa in a host biofilm infection model. Importantly, we detected compounds that inhibit c-di-GMP levels and showed significant influence on biofilm formation and virulence in P. aeruginosa in vitro and in vivo. Consequently, we offer proof-of-concept information regarding swift and practical drug screening assays, suitable for medium- to high-throughput applications, which target the c-di-GMP signaling pathways in this significant Gram-negative pathogen.

Bacterial biofilms are a major and ongoing concern for public health, featuring both inherited genetic resistance traits and a conferred innate tolerance to traditional antibiotic therapies. Consequently, there is a growing need for novel methods of drug delivery, to increase the efficacy of antimicrobial agents. This research evaluated the anti-biofilm and bactericidal effects of ultrasound responsive gas-microbubbles (MBs) of either air or nitric oxide, using an in vitro Pseudomonas aeruginosa biofilm model grown in artificial wound medium. The four lipid-based MB formulations evaluated were room-air MBs (RAMBs) and nitric oxide MBs (NOMBs) with no electrical charge, as well as cationic (+) RAMBs+ and NOMBs+. Two principal treatment conditions were used: i) ultrasound stimulated MBs only, and ii) ultrasound stimulated MBs with a sub-inhibitory concentration (4 µg/mL) of the antibiotic gentamicin. The total treatment time was divided into a 60 second passive MB interaction period prior to 40 second ultrasound exposure; each MB formulation was tested in triplicate. Ultrasound stimulated RAMBs and NOMBs without antibiotic achieved reductions in biofilm biomass of 93.3% and 94.0%, respectively. Their bactericidal efficacy however was limited, with a reduction in culturable cells of 26.9% and 65.3%, respectively. NOMBs with sub-inhibitory antibiotic produced the most significant reduction in biofilm biomass, corresponding to a 99.9% (SD ± 5.21%); and a 99.9% (SD ± 0.07%) (3-log) reduction in culturable bacterial cells. Cationic MBs were initially manufactured to promote binding of MBs to negatively charged biofilms, but these formulations also demonstrated intrinsic bactericidal properties. In the absence of antibiotic, the bactericidal efficacy of RAMB+ and NOMB+ was greater that of uncharged counterparts, reducing culturable cells by 84.7% and 86.1% respectively; increasing to 99.8% when combined with antibiotic. This study thus demonstrates the anti-biofilm and bactericidal utility of ultrasound stimulated MBs, and specifically is the first to demonstrate the efficacy of a NOMB for the dispersal and potentiation of antibiotics against bacterial biofilms in vitro. Importantly the biofilm system and complex growth-medium were selected to recapitulate key morphological features of in vivo biofilms. The results us offer new insight for the development of new clinical treatments, for example, in chronic wounds.

Current methods for diagnosing acute and complex infections mostly rely on culture-based methods and, for biofilms, fluorescence in-situ hybridization. These techniques are labor-intensive and can take 2-4 days to return a test result, especially considering an extra culturing step required for the antibiotic susceptibility testing (AST). This places a significant burden on healthcare providers, delaying treatment and leading to adverse patient outcomes. Here, we report the complementary use of our newly developed multi-excitation Raman spectroscopy (ME-RS) method with whole-genome sequencing (WGS). Four WHO priority pathogens are AST phenotyped and their antimicrobial resistance (AMR) profile determined by WGS. On application of ME-RS method we find high correlation with the WGS characterization. Highly accurate classification based on the species (98.93%), wild-type/non-wild type (99.45%), and presence or absence of thick peptidoglycan layers in cell walls (100%), as well as at the individual strain level (99.29%). These results clearly demonstrate the potential of ME-RS as a rapid and first-stage tool for species, resistance and strain-level classification which can be followed up by WGS for confirmation. Such a workflow can facilitate efficient antimicrobial stewardship to handle and prevent the spread of AMR.

The increasing awareness of the significance of microbial biofilms across different sectors is continuously revealing new areas of opportunity in the development of innovative technologies in translational research, which can address their detrimental effects, as well as exploit their benefits. Due to the extent of sectors affected by microbial biofilms, capturing their real financial impact has been difficult. This perspective highlights this impact globally, based on figures identified in a recent in-depth market analysis commissioned by the UK’s National Biofilms Innovation Centre (NBIC). The outputs from this analysis and the workshops organised by NBIC on its research strategic themes have revealed the breath of opportunities for translational research in microbial biofilms. However, there are still many outstanding scientific and technological challenges which must be addressed in order to catalyse these opportunities. This perspective discusses some of these challenges.