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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 Bacterial Adhesion and Biofilm Formation on Textured Breast Implant Shell Materials(2019-04) James, Garth A.; Boegli, Laura; Hancock, John; Bowersock, Lisa B.; Parker, Albert E.; Kinney, Brian M."Background Bacterial biofilms have been implicated with breast implant complications including capsular contracture and anaplastic large-cell lymphoma. The actual mechanisms for either are still under active investigation and are not clear. Due to their increased surface area, implants with textured surfaces may harbor greater biofilm loads than those with smooth surfaces. Methods Biofilm formation on the outer surface material was compared using implants with various surface areas and roughness, including Natrelle® (Smooth), SmoothSilk®/SilkSurface® (Silk), VelvetSurface ® (Velvet), Siltex®, and Biocell®. The roughness and surface area of each material were assessed using non-contact profilometry. Bacterial attachment (2 h) and biofilm formation (24 h) were evaluated for Staphylococcus epidermidis, Pseudomonas aeruginosa, and Ralstonia pickettii over nine independent experiments using a CDC biofilm reactor and viable plate counts (VPCs) as well as confocal scanning laser microscopy. VPCs of the textured implants were compared relative to the Smooth implant. Results Surface areas increased with roughness and were similar among the three least rough implants (Smooth, Silk, and Velvet) and among the roughest implants (Siltex and Biocell). Overall, VPC indicated there was significantly more bacterial attachment and biofilm formation on the Siltex and Biocell implants than the Silk or Velvet implants, although there were differences between species and time points. CSLM confirmed the formation of thicker biofilms on the implants with rougher surface textures. Conclusion This in vitro study confirmed that implant surfaces with rougher texture, resulting in more surface area, harbored greater biofilm loads than those with smoother surfaces.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 Evaluation of a 2-aminoimidazole variant as adjuvant treatment for dermal bacterial infections(2017-01) Draughn, G. Logan; Allen, C. Leigh; Routh, Patricia A.; Stone, Maria R.; Kirker, Kelly R.; Boegli, Laura; Schuchman, Ryan M.; Linder, Keith E.; Baynes, Ronald E.; James, Garth A.; Melander, Christian; Pollard, Angela; Cavanagh, John2-Aminoimidazole (2-AI)-based compounds have been shown to efficiently disrupt biofilm formation, disperse existing biofilms, and resensitize numerous multidrug-resistant bacteria to antibiotics. Using Pseudomonas aeruginosa and Staphylococcus aureus, we provide initial pharmacological studies regarding the application of a 2-AI as a topical adjuvant for persistent dermal infections. In vitro assays indicated that the 2-AI H10 is nonbactericidal, resensitizes bacteria to antibiotics, does not harm the integument, and promotes wound healing. Furthermore, in vivo application of H10 on swine skin caused no gross abnormalities or immune reactions. Taken together, these results indicate that H10 represents a promising lead dermal adjuvant compound.Item Development and application of a polymicrobial, in vitro, wound biofilm model(2012-03) Woods, J.; Boegli, Laura; Kirker, Kelly R.; Agostinho, Alessandra; Durch, Amanda M.; Pulcini, Elinor D.; Stewart, Philip S.; James, Garth A.Aims: The goal of this investigation was to develop an in vitro, polymicrobial, wound biofilm capable of supporting the growth of bacteria with variable oxygen requirements.Methods and Results: The strict anaerobe Clostridium perfringens was isolated by cultivating wound homogenates using the drip-flow reactor (DFR), and a three-species biofilm model was established using methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa and Cl. perfringens in the colony-drip-flow reactor model. Plate counts revealed that MRSA, Ps. aeruginosa and Cl. perfringens grew to 7·39 ± 0·45, 10·22 ± 0·22 and 7·13 ± 0.·77 log CFU per membrane, respectively. The three-species model was employed to evaluate the efficacy of two antimicrobial dressings, Curityâ„¢ AMD and Acticoatâ„¢, compared to sterile gauze controls. Microbial growth on Curityâ„¢ AMD and gauze was not significantly different, for any species, whereas Acticoatâ„¢ was found to significantly reduce growth for all three species.Conclusions: Using the colony-DFR, a three-species biofilm was successfully grown, and the biofilms displayed a unique structure consisting of distinct layers that appeared to be inhabited exclusively or predominantly by a single species.Significance and Impact of the Study: The primary accomplishment of this study was the isolation and growth of an obligate anaerobe in an in vitro model without establishing an artificially anaerobic environment.Item Anti-biofilm activity of silver nanoparticles against different microorganisms(2013-07) Martinez-Gutierrez, F.; Boegli, Laura; Agostinho, Alessandra; Sanchez, E. M.; Bach, H.; Ruiz, F.; James, Garth A.Biofilms confer protection from adverse environmental conditions and can be reservoirs for pathogenic organisms and sources of disease outbreaks, especially in medical devices. The goal of this research was to evaluate the anti-biofilm activities of silver nanoparticles (AgNPs) against several microorganisms of clinical interest. The antimicrobial activity of AgNPs was tested within biofilms generated under static conditions and also under high fluid shears conditions using a bioreactor. A 4-log reduction in the number of colony-forming units of Pseudomonas aeruginosa was recorded under turbulent fluid conditions in the CDC reactor on exposure to 100 mg ml 1 of AgNPs. The antibacterial activity of AgNPs on various microbial strains grown on polycarbonate membranes is reported. In conclusion, AgNPs effectively prevent the formation of biofilms and kill bacteria in established biofilms, which suggests that AgNPs could be used for prevention and treatment of biofilm-related infections. Further research and development are necessary to translate this technology into therapeutic and preventive strategies.Item Silver nanoparticles with antimicrobial activities against Streptococcus mutans and their cytotoxic effect(2015-10) Perez-Diaz, M. A.; Boegli, Laura; James, Garth A.; Velasquillo, C.; Sanchez-Sanchez, R.; Martinez-Martinez, R. E.; Martinez-Castanon, G. A.; Martinez-Gutierrez, F.Microbial resistance represents a challenge for the scientific community to develop new bioactive compounds. The goal of this research was to evaluate the antimicrobial activity of silver nanoparticles (AgNPs) against a clinical isolate of Streptococcus mutans, antibiofilm activity against mature S. mutans biofilms and the compatibility with human fibroblasts. The antimicrobial activity of AgNPs against the planktonic clinical isolate was size and concentration dependent, with smaller AgNPs having a lower minimum inhibitory concentration. A reduction of 2.3 log in the number of colony-forming units of S. mutans was observed when biofilms grown in a CDC reactor were exposed to 100 ppm of AgNPs of 9.5 ± 1.1 nm. However, AgNPs at high concentrations (> 10 ppm) showed a cytotoxic effect upon human dermal fibroblasts. AgNPs effectively inhibited the growth of a planktonic S. mutans clinical isolate and killed established S. mutans biofilms, which suggests that AgNPs could be used for prevention and treatment of dental caries. Further research and development are necessary to translate this technology into therapeutic and preventive strategies.Item Contribution of Stress Responses to Antibiotic Tolerance in Pseudomonas aeruginosa Biofilms(2015-04) Stewart, Philip S.; Franklin, Michael J.; Folsom, James P.; Boegli, Laura; James, Garth A.Enhanced tolerance of biofilm-associated bacteria to antibiotic treatments is likely due to a combination of factors, including changes in cell physiology as bacteria adapt to biofilm growth and the inherent physiological heterogeneity of biofilm bacteria. In this study, a transcriptomics approach was used to identify genes differentially expressed during biofilm growth of Pseudomonas aeruginosa. These genes were tested for statistically significant overlap, with independently compiled gene lists corresponding to stress responses and other putative antibiotic-protective mechanisms. Among the gene groups tested were those associated with biofilm response to tobramycin or ciprofloxacin, drug efflux pumps, acyl homoserine lactone quorum sensing, osmotic shock, heat shock, hypoxia stress, and stationary-phase growth. Regulons associated with Anr-mediated hypoxia stress, RpoS-regulated stationary-phase growth, and osmotic stress were significantly enriched in the set of genes induced in the biofilm. Mutant strains deficient in rpoS, relA and spoT, or anr were cultured in biofilms and challenged with ciprofloxacin and tobramycin. When challenged with ciprofloxacin, the mutant strain biofilms had 2.4- to 2.9-log reductions in viable cells compared to a 0.9-log reduction of the wild-type strain. Interestingly, none of the mutants exhibited a statistically significant alteration in tobramycin susceptibility compared to that with the wild-type biofilm. These results are consistent with a model in which multiple genes controlled by overlapping starvation or stress responses contribute to the protection of a P. aeruginosa biofilm from ciprofloxacin. A distinct and as yet undiscovered mechanism protects the biofilm bacteria from tobramycin.