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Item Critical analysis of methods to determine growth, control and analysis of biofilms for potential non-submerged antibiofilm surfaces and coatings(Elsevier BV, 2024-06) Redfern, J.; Cunliffe, A.J.; Goeres, D.M.; Azevedo, N.F.; Verran, J.The potential uses for antibiofilm surfaces reach across different sectors with significant resultant economic, societal and health impact. For those interested in using antibiofilm surfaces in the built environment, it is important that efficacy testing methods are relevant, reproducible and standardised where possible, to ensure data outputs are applicable to end-use, and comparable across the literature. Using pre-defined keywords, a review of literature reporting on antimicrobial surfaces (78 articles), within which a potential application was described as non-submerged/non-medical surface or coating with antibiofilm action, was undertaken. The most used methods utilized the growth of biofilm in submerged and static systems. Quantification varied (from most to least commonly used) across colony forming unit counts, non-microscopy fluorescence or spectroscopy, microscopy analysis, direct agar-contact, sequencing, and ELISA. Selection of growth media, microbial species, and incubation temperature also varied. In many cases, definitions of biofilm and attempts to quantify antibiofilm activity were absent or vague. Assessing a surface after biofilm recovery or assessing potential regrowth of a biofilm after initial analysis was almost entirely absent. It is clear the field would benefit from widely agreed and adopted approaches or guidance on how to select and incorporate end-use specific conditions, alongside minimum reporting guidelines may benefit the literature.Item Α,α-disubstituted β-amino amides eliminate Staphylococcus aureus biofilms by membrane disruption and biomass removal(Elsevier BV, 2023-12) Ausbacher, Dominik; Miller, Lindsey A.; Goeres, Darla M.; Stewart, Philip S.; Strøm, Morten B.; Fallarero, AdyaryBacterial biofilms account for up to 80% of all infections and complicate successful therapies due to their intrinsic tolerance to antibiotics. Biofilms also cause serious problems in the industrial sectors, for instance due to the deterioration of metals or microbial contamination of products. Efforts are put in finding novel strategies in both avoiding and fighting biofilms. Biofilm control is achieved by killing and/or removing biofilm or preventing transition to the biofilm lifestyle. Previous research reported on the anti-biofilm potency of α,α-disubstituted β-amino amides A1, A2 and A3, which are small antimicrobial peptidomimetics with a molecular weight below 500 Da. In the current study it was investigated if these derivatives cause a fast disintegration of biofilm bacteria and removal of Staphylococcus aureus biofilms. One hour incubation of biofilms with all three derivatives resulted in reduced metabolic activity and membrane permeabilization in S. aureus (ATCC 25923) biofilms. Bactericidal properties of these derivatives were attributed to a direct effect on membranes of biofilm bacteria. The green fluorescence protein expressing Staphylococcus aureus strain AH2547 was cultivated in a CDC biofilm reactor and utilized for disinfectant efficacy testing of A3, following the single tube method (American Society for Testing and Materials designation number E2871). A3 at a concentration of 90 μM acted as fast as 100 μM chlorhexidine and was equally effective. Confocal laser scanning microscopy studies showed that chlorhexidine treatment lead to fluorescence fading indicating membrane permeabilization but did not cause biomass removal. In contrast, A3 treatment caused a simultaneous biofilm fluorescence loss and biomass removal. These dual anti-biofilm properties make α,α-disubstituted β-amino amides promising scaffolds in finding new control strategies against recalcitrant biofilms.Item N-(Trimethylsilyl)-2-amino-5-nitrothiazole: An Efficient Reagent for the Direct Synthesis of 2-Amino-5-nitrothiazole-Based Antimicrobial Agents(Georg Thieme Verlag KG, 2022-11) Livinghouse, Tom; Koenig, Heidi N.; Demeritte, Amethyst R.; Nelson, Genevieve P.Here we report the synthesis of a novel reagent designed to prepare 2-amino-5-nitrothiazole (ANT) amides and analogues in high yields. N-(Trimethylsilyl)-2-amino-5-nitrothiazole (N-(TMS)-ANT) was prepared in 99% yield via silylation of ANT using 1,1,1,3,3,3-hexamethyldisilazane (HMDS), trimethylsilyl chloride (TMSCl), and catalytic saccharin. N-(TMS)-ANT is a superb reagent for the preparation of ANT amides in excellent yields. Notably, cyclic anhydrides and base-sensitive acyl chlorides can be utilized with N-(TMS)-ANT to furnish ANT amides that are difficult to prepare by previously reported procedures.Item Mitigation and use of biofilms in space for the benefit of human space exploration(Elsevier BV, 2023-12) Vélez Justiniano, Yo-Ann; Goeres, Darla M.; Sandvik, Elizabeth L.; Kjellerup, Birthe Veno; Sysoeva, Tatyana A.; Harris, Jacob S.; Warnat, Stephan; McGlennen, Matthew; Foreman, Christine M.; Yang, Jiseon; Li, Wenyan; Cassilly, Chelsi D.; Lott, Katelyn; HerrNeckar, Lauren E.Biofilms are self-organized communities of microorganisms that are encased in an extracellular polymeric matrix and often found attached to surfaces. Biofilms are widely present on Earth, often found in diverse and sometimes extreme environments. These microbial communities have been described as recalcitrant or protective when facing adversity and environmental exposures. On the International Space Station, biofilms were found in human-inhabited environments on a multitude of hardware surfaces. Moreover, studies have identified phenotypic and genetic changes in the microorganisms under microgravity conditions including changes in microbe surface colonization and pathogenicity traits. Lack of consistent research in microgravity-grown biofilms can lead to deficient understanding of altered microbial behavior in space. This could subsequently create problems in engineered systems or negatively impact human health on crewed spaceflights. It is especially relevant to long-term and remote space missions that will lack resupply and service. Conversely, biofilms are also known to benefit plant growth and are essential for human health (i.e., gut microbiome). Eventually, biofilms may be used to supply metabolic pathways that produce organic and inorganic components useful to sustaining life on celestial bodies beyond Earth. This article will explore what is currently known about biofilms in space and will identify gaps in the aerospace industry's knowledge that should be filled in order to mitigate or to leverage biofilms to the advantage of spaceflight.Item Sulfur cycling and host-virus interactions in Aquificales-dominated biofilms from Yellowstone’s hottest ecosystems(Springer Nature, 2021-10) McKay, Luke J.; Nigro, Olivia D.; Dlakić, Mensur; Luttrell, Karen M.; Rusch, Douglas B.; Fields, Matthew W.; Inskeep, William P.Modern linkages among magmatic, geochemical, and geobiological processes provide clues about the importance of thermophiles in the origin of biogeochemical cycles. The aim of this study was to identify the primary chemoautotrophs and host–virus interactions involved in microbial colonization and biogeochemical cycling at sublacustrine, vapor-dominated vents that represent the hottest measured ecosystems in Yellowstone National Park (~140 °C). Filamentous microbial communities exposed to extreme thermal and geochemical gradients were sampled using a remotely operated vehicle and subjected to random metagenome sequencing and microscopic analyses. Sulfurihydrogenibium (phylum Aquificae) was the predominant lineage (up to 84% relative abundance) detected at vents that discharged high levels of dissolved H2, H2S, and CO2. Metabolic analyses indicated carbon fixation by Sulfurihydrogenibium spp. was powered by the oxidation of reduced sulfur and H2, which provides organic carbon for heterotrophic community members. Highly variable Sulfurihydrogenibium genomes suggested the importance of intra-population diversity under extreme environmental and viral pressures. Numerous lytic viruses (primarily unclassified taxa) were associated with diverse archaea and bacteria in the vent community. Five circular dsDNA uncultivated virus genomes (UViGs) of ~40 kbp length were linked to the Sulfurihydrogenibium metagenome-assembled genome (MAG) by CRISPR spacer matches. Four UViGs contained consistent genome architecture and formed a monophyletic cluster with the recently proposed Pyrovirus genus within the Caudovirales. Sulfurihydrogenibium spp. also contained CRISPR arrays linked to plasmid DNA with genes for a novel type IV filament system and a highly expressed β-barrel porin. A diverse suite of transcribed secretion systems was consistent with direct microscopic analyses, which revealed an extensive extracellular matrix likely critical to community structure and function. We hypothesize these attributes are fundamental to the establishment and survival of microbial communities in highly turbulent, extreme-gradient environments.Item Search for a Shared Genetic or Biochemical Basis for Biofilm Tolerance to Antibiotics across Bacterial Species(American Society for Microbiology, 2022-04) Stewart, Philip S.; Williamson, Kerry S.; Boegli, Laura; Hamerly, Timothy; White, Ben; Scott, Liam; Hu, Xiao; Mumey, Brendan M.; Franklin, Michael J.; Bothner, Brian; Vital-Lopez, Francisco G.; Wallqvist, Anders; James, Garth A.Is there a universal genetically programmed defense providing tolerance to antibiotics when bacteria grow as biofilms? A comparison between biofilms of three different bacterial species by transcriptomic and metabolomic approaches uncovered no evidence of one. Single-species biofilms of three bacterial species (Pseudomonas aeruginosa, Staphylococcus aureus, and Acinetobacter baumannii) were grown in vitro for 3 days and then challenged with respective antibiotics (ciprofloxacin, daptomycin, and tigecycline) for an additional 24 h. All three microorganisms displayed reduced susceptibility in biofilms compared to planktonic cultures. Global transcriptomic profiling of gene expression comparing biofilm to planktonic and antibiotic-treated biofilm to untreated biofilm was performed. Extracellular metabolites were measured to characterize the utilization of carbon sources between biofilms, treated biofilms, and planktonic cells. While all three bacteria exhibited a species-specific signature of stationary phase, no conserved gene, gene set, or common functional pathway could be identified that changed consistently across the three microorganisms. Across the three species, glucose consumption was increased in biofilms compared to planktonic cells, and alanine and aspartic acid utilization were decreased in biofilms compared to planktonic cells. The reasons for these changes were not readily apparent in the transcriptomes. No common shift in the utilization pattern of carbon sources was discerned when comparing untreated to antibiotic-exposed biofilms. Overall, our measurements do not support the existence of a common genetic or biochemical basis for biofilm tolerance against antibiotics. Rather, there are likely myriad genes, proteins, and metabolic pathways that influence the physiological state of individual microorganisms in biofilms and contribute to antibiotic tolerance.Item Biofilms vs. cities and humans vs. aliens – a tale of reproducibility in biofilms(Elsevier BV, 2021-06) Azevedo, Nuno F.; Allkja, Jontana; Goeres, Darla M.In recent decades the scientific community has started to appreciate that most microorganisms live in complex 3D structures composed of cells, polysaccharides, and other components such as proteins, extracellular (e)DNA, and lipids. These structures are commonly designated 'biofilms'. Similar to other areas of research, biofilm studies have been affected by a lack of reproducibility. In this article, we propose a new scheme on how to classify the level of reproducibility in biofilms. This consists of four different levels: level 1, no reproducibility; level 2, standard reproducibility; level 3, potential standard reproducibility; and level 4, total reproducibility. Some methods aim to improve reproducibility by focusing on biofilm growth reactors, while others focus on biofilm characterization methods. Moreover, initiatives such as minimum information guidelines and biofilm-centered databases offer alternative strategies to tackle the reproducibility problem. The path to total reproducibility is certainly complex, but novel experimental and computational strategies are bringing us closer to achieving this goal.