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Item The Depletion Mechanism Actuates Bacterial Aggregation by Exopolysaccharides and Determines Species Distribution & Composition in Bacterial Aggregates(Frontiers Media SA, 2022-06) Secor, Patrick R.; Michaels, Lia A.; Bublitz, DeAnna C.; Jennings, Laura K.; Singh, Pradeep K.Bacteria in natural environments and infections are often found in cell aggregates suspended in polymer-rich solutions, and aggregation can promote bacterial survival and stress resistance. One aggregation mechanism, called depletion aggregation, is driven by physical forces between bacteria and high concentrations of polymers in the environment rather than bacterial activity per se. As such, bacteria aggregated by the depletion mechanism will disperse when polymer concentrations fall unless other adhesion mechanisms supervene. Here we investigated whether the depletion mechanism can actuate the aggregating effects of Pseudomonas aeruginosa exopolysaccharides for suspended (i.e. not surface attached) bacteria, and how depletion affects bacterial inter-species interactions. We found that cells overexpressing the exopolysaccharides Pel and Psl remained aggregated after short periods of depletion aggregation whereas wild-type and mucoid P. aeruginosa did not. In co-culture, depletion aggregation had contrasting effects on P. aeruginosa’s interactions with coccus- and rod-shaped bacteria. Depletion caused S. aureus (cocci) and P. aeruginosa (rods) to segregate from each other and S. aureus to resist secreted P. aeruginosa antimicrobial factors resulting in species co-existence. In contrast, depletion aggregation caused P. aeruginosa and Burkholderia sp. (both rods) to intermix, enhancing type VI secretion inhibition of Burkholderia by P. aeruginosa, leading to P. aeruginosa dominance. These results show that in addition to being a primary cause of aggregation in polymer-rich suspensions, physical forces inherent to the depletion mechanism can promote aggregation by some self-produced exopolysaccharides and determine species distribution and composition of bacterial communities.Item Clothing Textiles as Carriers of Biological Ice Nucleation Active Particles(American Chemical Society, 2024-03) Teska, Christy J.; Dieser, Markus; Foreman, Christine M.Microplastics have littered the globe, with synthetic fibers being the largest source of atmospheric microplastics. Many atmospheric particles can act as ice nucleators, thereby affecting the microphysical and radiative properties of clouds and, hence, the radiative balance of the Earth. The present study focused on the ice-nucleating ability of fibers from clothing textiles (CTs), which are commonly shed from the normal wear of apparel items. Results from immersion ice nucleation experiments showed that CTs were effective ice nucleators active from −6 to −12 °C, similar to common biological ice nucleators. However, subsequent lysozyme and hydrogen peroxide digestion stripped the ice nucleation properties of CTs, indicating that ice nucleation was biological in origin. Microscopy confirmed the presence of biofilms (i.e., microbial cells attached to a surface and enclosed in an extracellular polysaccharide matrix) on CTs. If present in sufficient quantities in the atmosphere, biological particles (biofilms) attached to fibrous materials could contribute significantly to atmospheric ice nucleation.Item Pseudomonas aeruginosa aggregates in cystic fibrosis sputum produce exopolysaccharides that likely impede current therapies(Elsevier BV, 2021-02) Jennings, Laura K.; Dreifus, Julia E.; Reichhardt, Courtney; Storek, Kelly M.; Secor, Patrick R.; Wozniak, Daniel J.; Hisert, Katherine B.; Parsek, Matthew R.In cystic fibrosis (CF) airways, Pseudomonas aeruginosa forms cellular aggregates called biofilms that are thought to contribute to chronic infection. To form aggregates, P. aeruginosa can use different mechanisms, each with its own pathogenic implications. However, how they form in vivo is controversial and unclear. One mechanism involves a bacterially produced extracellular matrix that holds the aggregates together. Pel and Psl exopolysaccharides are structural and protective components of this matrix. We develop an immunohistochemical method to visualize Pel and Psl in CF sputum. We demonstrate that both exopolysaccharides are expressed in the CF airways and that the morphology of aggregates is consistent with an exopolysaccharide-dependent aggregation mechanism. We reason that the cationic exopolysaccharide Pel may interact with some of the abundant anionic host polymers in sputum. We show that Pel binds extracellular DNA (eDNA) and that this interaction likely impacts current therapies by increasing antimicrobial tolerance and protecting eDNA from digestion.Item Exopolysaccharide biosynthetic glycoside hydrolases can be utilized to disrupt and prevent Pseudomonas aeruginosa biofilms(American Association for the Advancement of Science, 2016-05) Baker, Perrin; Hill, Preston J.; Snarr, Brendan D.; Alnabelseya, Noor; Pestrak, Matthew J.; Lee, Mark J.; Jennings, Laura K.; Tam, John; Melnyk, Roman A.; Parsek, Matthew R.; Sheppard, Donald C.; Wozniak, Daniel J.; Howell, P. LynneBacterial biofilms present a significant medical challenge because they are recalcitrant to current therapeutic regimes. A key component of biofilm formation in the opportunistic human pathogen Pseudomonas aeruginosa is the biosynthesis of the exopolysaccharides Pel and Psl, which are involved in the formation and maintenance of the structural biofilm scaffold and protection against antimicrobials and host defenses. Given that the glycoside hydrolases PelAh and PslGh encoded in the pel and psl biosynthetic operons, respectively, are utilized for in vivo exopolysaccharide processing, we reasoned that these would provide specificity to target P. aeruginosa biofilms. Evaluating these enzymes as potential therapeutics, we demonstrate that these glycoside hydrolases selectively target and degrade the exopolysaccharide component of the biofilm matrix. PelAh and PslGh inhibit biofilm formation over a 24-hour period with a half maximal effective concentration (EC50) of 69.3 ± 1.2 and 4.1 ± 1.1 nM, respectively, and are capable of disrupting preexisting biofilms in 1 hour with EC50 of 35.7 ± 1.1 and 12.9 ± 1.1 nM, respectively. This treatment was effective against clinical and environmental P. aeruginosa isolates and reduced biofilm biomass by 58 to 94%. These noncytotoxic enzymes potentiated antibiotics because the addition of either enzyme to a sublethal concentration of colistin reduced viable bacterial counts by 2.5 orders of magnitude when used either prophylactically or on established 24-hour biofilms. In addition, PelAh was able to increase neutrophil killing by ~50%. This work illustrates the feasibility and benefits of using bacterial exopolysaccharide biosynthetic glycoside hydrolases to develop novel antibiofilm therapeutics.Item Biofilm assembly becomes crystal clear – filamentous bacteriophage organize the Pseudomonas aeruginosa biofilm matrix into a liquid crystal(Shared Science Publishers OG, 2016-01) Patrick R. Secor; Laura K. Jennings; Lia A. Michaels; Johanna M. Sweere; Pradeep K. Singh; William C. Parks; Paul L. BollykyPseudomonas aeruginosa is an opportunistic bacterial pathogen associated with many types of chronic infection. At sites of chronic infection, such as the airways of people with cystic fibrosis (CF), P. aeruginosa forms biofilm-like aggregates. These are clusters of bacterial cells encased in a polymer-rich matrix that shields bacteria from environmental stresses and antibiotic treatment. When P. aeruginosa forms a biofilm, large amounts of filamentous Pf bacteriophage (phage) are produced. Unlike most phage that typically lyse and kill their bacterial hosts, filamentous phage of the genus Inovirus, which includes Pf phage, often do not, and instead are continuously extruded from the bacteria. Here, we discuss the implications of the accumulation of filamentous Pf phage in the biofilm matrix, where they interact with matrix polymers to organize the biofilm into a highly ordered liquid crystal. This structural configuration promotes bacterial adhesion, desiccation survival, and antibiotic tolerance – all features typically associated with biofilms. We propose that Pf phage make structural contributions to P. aeruginosa biofilms and that this constitutes a novel form of symbiosis between bacteria and bacteriophage.Item Entropically driven aggregation of bacteria by host polymers promotes antibiotic tolerance in Pseudomonas aeruginosa(Proceedings of the National Academy of Sciences, 2018-10) Secor, Patrick R.; Michaels, Lia A.; Ratjen, Anina; Jennings, Laura K.; Singh, Pradeep K.Bacteria causing chronic infections are generally observed living in cell aggregates suspended in polymer-rich host secretions, and bacterial phenotypes induced by aggregated growth may be key factors in chronic infection pathogenesis. Bacterial aggregation is commonly thought of as a consequence of biofilm formation; however the mechanisms producing aggregation in vivo remain unclear. Here we show that polymers that are abundant at chronic infection sites cause bacteria to aggregate by the depletion aggregation mechanism, which does not require biofilm formation functions. Depletion aggregation is mediated by entropic forces between uncharged or like-charged polymers and particles (e.g., bacteria). Our experiments also indicate that depletion aggregation of bacteria induces marked antibiotic tolerance that was dependent on the SOS response, a stress response activated by genotoxic stress. These findings raise the possibility that targeting conditions that promote depletion aggregation or mechanisms of depletion-mediated tolerance could lead to new therapeutic approaches to combat chronic bacterial infections.Item Anti-Biofilm Efficacy of Commonly Used Wound Care Products in In Vitro Settings(MDPI AG, 2023-03) Regulski, Matthew; Myntti, Matthew F.; James, Garth A.Considering the prevalence and pathogenicity of biofilms in wounds, this study was designed to evaluate the anti-biofilm capabilities of eight commercially available wound care products using established in vitro assays for biofilms. The products evaluated included dressings with multiple delivery formats for ionic silver including nanocrystalline, gelling fibers, polyurethane (PU) foam, and polymer matrix. Additionally, non-silver-based products including an extracellular polymeric substance (EPS)-dissolving antimicrobial wound gel (BDWG), a collagenase-based debriding ointment and a fish skin-based skin substitute were also evaluated. The products were evaluated on Staphylococcus aureus and Pseudomonas aeruginosa mixed-species biofilms grown using colony drip flow reactor (CDFR) and standard drip flow reactor (DFR) methodologies. Anti-biofilm efficacy was measured by viable plate counts and confocal scanning laser microscopy (CSLM). Four of the eight wound care products tested were efficacious in inhibiting growth of new biofilm when compared with untreated controls. These four products were further evaluated against mature biofilms. BDWG was the only product that achieved greater than 2-log growth reduction (5.88 and 6.58 for S. aureus and P. aeruginosa, respectively) of a mature biofilm. Evaluating both biofilm prevention and mature biofilm disruption capacity is important to a comprehensive understanding of the anti-biofilm efficacy of wound care products.Item Antimicrobial effects of an acidified nitrite foam on drip flow reactor biofilm(European Wound Management Association, 2024-04) Miller, C. Michael; James, Garth; Bell, David; Schultz, GregBackground. Nitric oxide (NO) plays critical roles in wound healing, including stimulating vasodilation, angiogenesis and broad antimicrobial activity. Aim. To measure the effect of an acidified nitrite foam (ANF) on biofilms created by six different microbes. Methods. A novel method of generating, delivering and topically applying NO gas at the point of care was developed using ANF in a mixed bubble foam and was tested in vitro against six common microbial wound pathogens. Results. A single 5-minute topical exposure of the NO bubble gas formulation generated a 5.8-log10 reduction of mature biofilm of Pseudomonas aeruginosa, a 5.1-log10 reduction of Staphylococcus aureus biofilm, a 4.0-log10 reduction of Staphylococcus epidermidis biofilm, a 3.2-log10 reduction of Proteus mirabilis biofilm, a 2.7-log10 reduction of Acinetobacter baumannii biofilm, and a 1.5-log10 reduction of Candida albicans biofilm. Conclusion. The efficacy of a 5-minute treatment of ANF used on biofilms of P. aeruginosa, A. baumannii, S. aureus, C. albicans, P. mirabilis and S. epidermidis was confirmed. The treatment resulted in a significant reduction in colony-forming units per square centimetre (CFU/cm2) comparable to or surpassing other methods of NO gas application, suggesting NO containing foam’s utility as a point of care solution for chronic wounds with elevated bioburden and biofilms where levels of endogenously produced NO may be insufficient for wound healing completion.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 Development of Martian saline seep models and their implications for planetary protection(Elsevier BV, 2023-12) Mettler, Madelyn K.; Goemann, Hannah M.; Mueller, Rebecca C.; Vanegas, Oscar A.; Lopez, Gabriela; Singh, Nitin; Venkateswaran, Kasthuri; Peyton, Brent M.While life on Mars has not been found, Earth-based microorganisms may contaminate the Red Planet during rover expeditions and human exploration. Due to the survival advantages conferred by the biofilm morphology to microorganisms, such as resistance to UV and osmotic stress, biofilms are particularly concerning from a planetary protection perspective. Modeling and data from the NASA Phoenix mission indicate that temporary liquid water might exist on Mars in the form of high salinity brines. These brines could provide colonization opportunities for terrestrial microorganisms brought by spacecraft or humans. To begin testing for potential establishment of microbes, results are presented from a simplified laboratory model of a Martian saline seep inoculated with sediment from Hailstone Basin, a terrestrial saline seep in Montana (USA). The seep was modeled as a sand-packed drip flow reactor at room temperature fed media with either 1 M MgSO4 or 1 M NaCl. Biofilms were established within the first sampling point of each experiment. Endpoint 16S rRNA gene community analysis showed significant selection of halophilic microorganisms by the media. Additionally, we detected 16S rRNA gene sequences highly similar to microorganisms previously detected in two spacecraft assembly cleanrooms. These experimental models provide an important foundation for identifying microbes that could hitch-hike on spacecraft and may be able to colonize Martian saline seeps. Future model optimization will be vital to informing cleanroom sterilization procedures.