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Item Simulation of catalase-dependent tolerance of microbial biofilm to hydrogen peroxide with a biofilm computer model(Springer Science and Business Media LLC, 2023-08) Stewart, Philip S.; Owkes, MarkHydrogen peroxide (HP) is a common disinfectant and antiseptic. When applied to a biofilm, it may be expected that the top layer of the biofilm would be killed by HP, the HP would penetrate further, and eventually eradicate the entire biofilm. However, using the Biofilm.jl computer model, we demonstrate a mechanism by which the biofilm can persist, and even become thicker, in the indefinite treatment with an HP solution at concentrations that are lethal to planktonic microorganisms. This surprising result is found to be dependent on the neutralization of HP by dead biomass, which provides protection for living biomass deeper within the biofilm. Practically, to control a biofilm, this result leads to the concept of treating with an HP dose exceeding a critical threshold concentration rather than a sustained, lower-concentration treatment.Item Experimental Designs to Study the Aggregation and Colonization of Biofilms by Video Microscopy With Statistical Confidenc(Frontiers Media SA, 2022-01) Pettygrove, Brian A.; Smith, Heidi J.; Pallister, Kyler B.; Voyich, Jovanka M.; Stewart, Philip S.; Parker, Albert E.The goal of this study was to quantify the variability of confocal laser scanning microscopy (CLSM) time-lapse images of early colonizing biofilms to aid in the design of future imaging experiments. To accomplish this a large imaging dataset consisting of 16 independent CLSM microscopy experiments was leveraged. These experiments were designed to study interactions between human neutrophils and single cells or aggregates of Staphylococcus aureus (S. aureus) during the initial stages of biofilm formation. Results suggest that in untreated control experiments, variability differed substantially between growth phases (i.e., lag or exponential). When studying the effect of an antimicrobial treatment (in this case, neutrophil challenge), regardless of the inoculation level or of growth phase, variability changed as a frown-shaped function of treatment efficacy (i.e., the reduction in biofilm surface coverage). These findings were used to predict the best experimental designs for future imaging studies of early biofilms by considering differing (i) numbers of independent experiments; (ii) numbers of fields of view (FOV) per experiment; and (iii) frame capture rates per hour. A spreadsheet capable of assessing any user-specified design is included that requires the expected mean log reduction and variance components from user-generated experimental results. The methodology outlined in this study can assist researchers in designing their CLSM studies of antimicrobial treatments with a high level of statistical confidence.Item Sulfenate Esters of Simple Phenols Exhibit Enhanced Activity against Biofilms(American Chemical Society, 2020-03) Walsh, Danica J.; Livinghouse, Tom; Durling, Greg M.; Chase-Bayless, Yenny; Arnold, Adrienne D.; Stewart, Philip S.The recalcitrance exhibited by microbial biofilms to conventional disinfectants has motivated the development of new chemical strategies to control and eradicate biofilms. The activities of several small phenolic compounds and their trichloromethylsulfenyl ester derivatives were evaluated against planktonic cells and mature biofilms of Staphylococcus epidermidis and Pseudomonas aeruginosa. Some of the phenolic parent compounds are well-studied constituents of plant essential oils, for example, eugenol, menthol, carvacrol, and thymol. The potency of sulfenate ester derivatives was markedly and consistently increased toward both planktonic cells and biofilms. The mean fold difference between the parent and derivative minimum inhibitory concentration against planktonic cells was 44 for S. epidermidis and 16 for P. aeruginosa. The mean fold difference between the parent and derivative biofilm eradication concentration for 22 tested compounds against both S. epidermidis and P. aeruginosa was 3. This work demonstrates the possibilities of a new class of biofilm-targeting disinfectants deploying a sulfenate ester functional group to increase the antimicrobial potency toward microorganisms in biofilms.Item Novel phenolic antimicrobials enhanced activity of iminodiacetate prodrugs against biofilm and planktonic bacteria(Wiley, 2020-09) Walsh, Danica J.; Livinghouse, Tom; Durling, Greg M.; Arnold, Adrienne D.; Brasier, Whitney; Berry, Luke; Goeres, Darla M.; Stewart, Philip S.Prodrugs are pharmacologically attenuated derivatives of drugs that undergo bioconversion into the active compound once reaching the targeted site, thereby maximizing their efficiency. This strategy has been implemented in pharmaceuticals to overcome obstacles related to absorption, distribution, and metabolism, as well as with intracellular dyes to ensure concentration within cells. In this study, we provide the first examples of a prodrug strategy that can be applied to simple phenolic antimicrobials to increase their potency against mature biofilms. The addition of (acetoxy)methyl iminodiacetate groups increases the otherwise modest potency of simple phenols. Biofilm-forming bacteria exhibit a heightened tolerance toward antimicrobial agents, thereby accentuating the need for new antibiotics as well as those, which incorporate novel delivery strategies to enhance activity toward biofilms.Item Delayed neutrophil recruitment allows nascent Staphylococcus aureus biofilm formation and immune evasion(Elsevier BV, 2021-08) Pettygrove, Brian A.; Kratofil, Rachel M.; Alhede, Maria; Jensen, Peter O.; Newton, MIchelle; Qvortup, Klaus; Pallister, Kyler B.; Bjarnsholt, Thomas; Kubes, Paul; Voyich, Jovanka M.; Stewart, Philip S.Biofilms that form on implanted medical devices cause recalcitrant infections. The early events enabling contaminating bacteria to evade immune clearance, before a mature biofilm is established, are poorly understood. Live imaging in vitro demonstrated that Staphylococcus aureus sparsely inoculated on an abiotic surface can go undiscovered by human neutrophils, grow, and form aggregates. Small (~50 μm2) aggregates of attached bacteria resisted killing by human neutrophils, resulting in neutrophil lysis and bacterial persistence. In vivo, neutrophil recruitment to a peritoneal implant was spatially heterogenous, with some bacterial aggregates remaining undiscovered by neutrophils after 24 hours. Intravital imaging in mouse skin revealed that attached S. aureus aggregates grew and remained undiscovered by neutrophils for up to three hours. These results suggest a model in which delayed recruitment of neutrophils to an abiotic implant presents a critical window in which bacteria establish a nascent biofilm and acquire tolerance to neutrophil killing.Item Nisin penetration and efficacy against Staphylococcus aureus biofilms under continuous-flow conditions(2019-07) Godoy-Santos, Fernanda; Pitts, Betsey; Stewart, Philip S.; Mantovani, Hilario C.Biofilms may enhance the tolerance of bacterial pathogens to disinfectants, biocides and other stressors by restricting the penetration of antimicrobials into the matrix-enclosed cell aggregates, which contributes to the recalcitrance of biofilm-associated infections. In this work, we performed real-time monitoring of the penetration of nisin into the interior of Staphylococcus aureus biofilms under continuous flow and compared the efficacy of this lantibiotic against planktonic and sessile cells of S. aureus . Biofilms were grown in Center for Disease Control (CDC) reactors and the spatial and temporal effects of nisin action on S. aureus cells were monitored by real-time confocal microscopy. Under continuous flow, nisin caused loss of membrane integrity of sessile cells and reached the bottom of the biofilms within ~20 min of exposure. Viability analysis using propidium iodide staining indicated that nisin was bactericidal against S. aureus biofilm cells. Time-kill assays showed that S. aureus viability reduced 6.71 and 1.64 log c.f.u. ml-1 for homogenized planktonic cells in exponential and stationary phase, respectively. For the homogenized and intact S. aureus CDC biofilms, mean viability decreased 1.25 and 0.50 log c.f.u. ml-1, respectively. Our results demonstrate the kinetics of biofilm killing by nisin under continuous-flow conditions, and shows that alterations in the physiology of S. aureus cells contribute to variations in sensitivity to the lantibiotic. The approach developed here could be useful to evaluate the antibiofilm efficacy of other bacteriocins either independently or in combination with other antimicrobials.Item Analysis of Clostridium difficile biofilms: imaging and antimicrobial treatment(2018-01) James, Garth A.; Chesnel, L.; Boegli, Laura; Pulcini, Elinor D.; Fisher, Steve T.; Stewart, Philip S.BACKGROUND: Clostridium difficile, a spore-forming Gram-positive anaerobic bacillus, is the most common causative agent of healthcare-associated diarrhoea. Formation of biofilms may protect C. difficile against antibiotics, potentially leading to treatment failure. Furthermore, bacterial spores or vegetative cells may linger in biofilms in the gut causing C. difficile infection recurrence. OBJECTIVES: In this study, we evaluated and compared the efficacy of four antibiotics (fidaxomicin, surotomycin, vancomycin and metronidazole) in penetrating C. difficile biofilms and killing vegetative cells. METHODS: C. difficile biofilms grown initially for 48 or 72 h using the colony biofilm model were then treated with antibiotics at a concentration of 25 × MIC for 24 h. Vegetative cells and spores were enumerated. The effect of treatment on biofilm structure was studied by scanning electron microscopy (SEM). The ability of fidaxomicin and surotomycin to penetrate biofilms was studied using fluorescently tagged antibiotics. RESULTS: Both surotomycin and fidaxomicin were significantly more effective than vancomycin or metronidazole (P < 0.001) at killing vegetative cells in established biofilms. Fidaxomicin was more effective than metronidazole at reducing viable spore counts in biofilms (P < 0.05). Fluorescently labelled surotomycin and fidaxomicin penetrated C. difficile biofilms in < 1 h. After 24 h of treatment, SEM demonstrated that both fidaxomicin and surotomycin disrupted the biofilm structure, while metronidazole had no observable effect. CONCLUSIONS: Fidaxomicin is effective in disrupting C. difficile biofilms, killing vegetative cells and decreasing spore counts.Item Paired methods to measure biofilm killing and removal: a case study with Penicillin G treatment of Staphylococcus aureus biofilm(2018-03) Ausbacher, D.; Lorenz, Lindsey A.; Pitts, Betsey; Stewart, Philip S.; Goeres, Darla M.Biofilms are microbial aggregates that show high tolerance to antibiotic treatments in vitro and in vivo. Killing and removal are both important in biofilm control, therefore methods that measure these two mechanisms were evaluated in a parallel experimental design. Kill was measured using the single tube method (ASTM method E2871) and removal was determined by video microscopy and image analysis using a new treatment flow cell. The advantage of the parallel test design is that both methods used biofilm covered coupons harvested from a CDC biofilm reactor, a well-established and standardized biofilm growth method. The control Staphylococcus aureus biofilms treated with growth medium increased by 0 6 logs during a 3-h contact time. Efficacy testing showed biofilms exposed to 400 lmol l1 penicillin G decreased by only 0 3 logs. Interestingly, time-lapse confocal scanning laser microscopy revealed that penicillin G treatment dispersed the biofilm despite being an ineffective killing agent. In addition, no biofilm removal was detected when assays were performed in 96-well plates. These results illustrate that biofilm behaviour and impact of treatments can vary substantially when assayed by different methods. Measuring both killing and removal with well-characterized methods will be crucial for the discovery of new anti-biofilm strategies.Item Bacterial biofilms: A common cause of persistent infections(1999-05) Costerton, J. William; Stewart, Philip S.; Greenberg, E. P.Bacteria that attach to surfaces aggregate in a hydrated polymeric matrix of their own synthesis to form biofilms. Formation of these sessile communities and their inherent resistance to antimicrobial agents are at the root of many persistent and chronic bacterial infections. Studies of biofilms have revealed differentiated, structured groups of cells with community properties. Recent advances in our understanding of the genetic and molecular basis of bacterial community behavior point to therapeutic targets that may provide a means for the control of biofilm infections.Item Electrolytic generation of oxygen partially explains electrical enhancement of tobramycin efficacy against pseudomonas aeruginosa biofilm(1999-02) Stewart, Philip S.; Wattanakaroon, Wanida; Goodrum, L.; Fortun, Susana M.; McLeod, Bruce R.The role of electrolysis products, including protons, hydroxyl ions, reactive oxygen intermediates, oxygen, hydrogen, and heat, in mediating electrical enhancement of killing of Pseudomonas aeruginosa biofilms by tobramycin (the bioelectric effect) was investigated. The log reduction in biofilm viable cell numbers compared to the numbers for the untreated positive control effected by antibiotic increased from 2.88 in the absence of electric current to 5.58 in the presence of electric current. No enhancement of antibiotic efficacy was observed when the buffer composition was changed to simulate the reduced pH that prevails during electrolysis. Neither did stabilization of the pH during electrical treatment by increasing the buffer strength eliminate the bioelectric effect. The temperature increase measured in our experiments, less than 0.2°C, was far too small to account for the greatly enhanced antibiotic efficacy. The addition of sodium thiosulfate, an agent capable of rapidly neutralizing reactive oxygen intermediates, did not abolish electrical enhancement of killing. The bioelectric effect persisted when all of the ionic constituents of the medium except the two phosphate buffer components were omitted. This renders the possibility of electrochemical generation of an inhibitory ion, such as nitrite from nitrate, an unlikely explanation for electrical enhancement. The one plausible explanation for the bioelectric effect revealed by this study was the increased delivery of oxygen to the biofilm due to electrolysis. When gaseous oxygen was bubbled into the treatment chamber during exposure to tobramycin (without electric current), a 1.8-log enhancement of killing resulted. The enhancement of antibiotic killing by oxygen was not due simply to physical disturbances caused by sparging the gas because similar delivery of gaseous hydrogen caused no enhancement whatsoever.