Browsing by Subject "Biofilms"
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Item Biofilm Formation by Moraxella Catarrhalis(2004-05-04) Pearson, Melanie Michelle; Hansen, Eric J.This is the first detailed study of biofilm formation in vitro by the Gram-negative bacterial pathogen Moraxella catarrhalis. Growth of M. catarrhalis in a continuous-culture biofilm system resulted in little detectable change in outer membrane protein production compared to broth-grown M. catarrhalis. Biofilm-grown M. catarrhalis may produce an extracellular polysaccharide and different colony phenotypes when grown in this continuous culture biofilm system. Transmission electron microscopy of biofilm-grown M. catarrhalis cells revealed abundant projections extending from the bacterial cell surface that were identified as the proteins UspA1, UspA2, and Hag, which are all putative members of the autotransporter protein family. The Hag protein of strain O35E was shown to be necessary for hemagglutination, autoagglutination, and binding of human IgD. A crystal violet-based assay utilizing 24-well tissue culture plates was also used to evaluate biofilm formation by M. catarrhalis. The ability of M. catarrhalis strains to form biofilms in this crystal violet-based assay varied considerably, but most strains form little or no biofilm in this system. Screening of M. catarrhalis transposon insertion mutants using the crystal violet-based assay revealed that the UspA1 or the related UspA2H proteins play a strain-dependent, positive role in biofilm formation. Expression of the Hag protein prevented biofilm formation in tissue culture plates by several M. catarrhalis strains. In contrast, there appears to be a positive selection for Hag expression by strain O46E in the continuous-culture biofilm system. Proteins involved in cell wall recycling may also be involved in biofilm formation by M. catarrhalis. Nucleotide sequence analysis, site-directed mutagenesis, and domain swapping experiments indicated that the N-terminal region of UspA1 or UspA2H is likely involved in biofilm formation by M. catarrhalis.Item Biofilms in obstructive lung disease(2004-06-24) Weissler, Jonathan C.Item Efficacy of Contact Lens Care Solutions Against Neutrophil Enhanced Biofilms(2015-01-26) Hinojosa, Jorge A.; Patel, Naiya; Zhu, Meifang; Robertson, Danielle M.PURPOSE: To evaluate currently available chemically preserved and peroxide-based lens care products (LCPs) antimicrobial efficacies against neutrophil enhanced biofilms in reference strains of S.auerus(SA), S. marcescens (SM), S. maltophilia(ST), and P.aeruginosa(PA). METHODS: Lotrafilcon B lenses were inoculated with 2.5 x 107 CFUs of American Type Culture Collection (ATCC) reference strains of either SA, SM, ST, or PA and 8.3x106 cells of human neutrophils harvested by whole blood centrifugation. Lenses were then incubated over night under conditions that facilitate biofilm formation. Lenses were then disinfected with either multipurpose contact lens solutions (MPS): Biotrue (BT), PureMoist (PM) or hydrogen peroxide-based lens care systems (HPB): ClearCare (CC), PeroxiClear (PC) according to specific manufacture guidelines. Antimicrobial activity was then quantified by quantitative culturing (colony-forming units) and by fluorescence confocal microscopy (FCM) using LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes, Eugene, OR). RESULTS: Mean colony growth counts for each bacterial strain + neutrophils were: SA= 1.44 x 107 (CFU/ml), SM= 2.56x109 (CFU/ml), ST= 1.17x1010 (CFU/ml), PA= 3.43x109 (CFU/ml). After treatment, SA exhibited a 7-log reduction with all LCPs (P=0.609); SM exhibited 9-log reduction for all LCPs except BT, which showed a 7-log reduction (P < 0.001); ST exhibited a 9-log to 10-log reduction for all LCPs (P=0.040); PA showed a 9-log reduction for all LCPs (P=1.000). FCM with viability staining revealed the presence many unviable bacteria still adhered to the lens surface after treatment with LCPs. CONCLUSIONS: Although all LCPs meet FDA criterion of a 3-log reduction minimum even against neutrophil enhanced biofilms, FCM revealed that many remnants still remain adhered on the lens surface after just one use. Further deposition of bacterial products from continued used could enhance biofilm formation and promote a host inflammatory response, both of which could precipitate into a corneal infiltrative event (CIE). In addition, mean colony growth counts showed that SA exhibited the lowest growth on lens surfaces, which may explain why SA is more commonly associated with sterile CIEs.Item Employing Alternating Magnetic Fields for Biofilm Destruction(2020-01-21) Vachon, M. Jonathan; Wang, Qi; Pybus, Christine; Shaikh, Sumbul; Chopra, Rajiv; Greenberg, DavidBACKGROUND: Prosthetic Joint Infections (PJI) are a common complication of implant surgery. Due to biofilm formation, treatment is costly, includes weeks of antibiotic therapy, and even total replacement of the prosthesis. However, a non-invasive thermal method of biofilm eradication has recently been developed: using high-frequency alternating magnetic fields (AMF) to destroy biofilm via induction. METHODS: The experiment used Staphylococcus aureus, a prototypic pathogen implicated in PJI. Stainless steel rings were used to mimic prosthetic joints. Biofilms of Methicillin-Sensitive Staphylococcus aureus (UAMS1-lux) were grown on stainless steel rings in a shaking incubator for 24 hours, 110rpm at 37°C, in Tryptic Soy Broth media. The rings were then resuspended in fresh media and incubated for another 24 hours at 110rpm, 37°C. Untreated rings, Antibiotics only, AMF only, and a combination of AMF + Abx were tested. Biofilms in the latter 2 categories underwent 3s pulses of AMF exposure every 5 minutes for 15, 30, or 60 minutes at a target temperature of 65°C every 12 hours for 24 hours. Ceftriaxone (2.0μg/ml) was used for the Abx conditions. Rings were sonicated at the indicated timepoints and colony-forming units (CFU) were determined. RESULTS: A synergistic effect between AMF and antibiotics was seen. At 12 hours, the Abx only and AMF only treatments showed regrowth; however, the combination therapy showed a 2.1-log decrease in biofilm CFU. Similarly, at 24 hours, solo AMF treatment showed total regrowth and Abx only treatment showed modest bactericidal effects (2.1 log reduction). However, combination therapy at 24hr showed a 5.35 log reduction and reached the limit of detection of the assay. Additionally, we are investigating the effects of AMF with Linezolid (2.0 μg/ml). At 24hrs, a 4.3 log reduction in biofilm CFU was observed in the combination treatment, while solo treatments showed total regrowth. CONCLUSIONS: These in vitro results serve as a strong basis for future work on AMF utilization in treatment of PJI. AMF and antibiotics are synergistic in reducing biofilm off metal. The observed bactericidal effects combined with this non-invasive means have wide and significant implications in improving the patient's quality of life as well as improving healthcare costs of PJI treatment.Item Non-Invasive Eradication of Biofilm on Metal Implants Using Alternating Magnetic Fields (AMF) and Antibiotics(August 2021) Wang, Qi; Madhuranthakam, Ananth; Chopra, Rajiv; Greenberg, David; Lewis, Matthew Allen; Fiolka, RetoHundreds of thousands of human implant procedures require surgical revision each year due to infection. Infections are difficult to treat with conventional antibiotics due to the formation of biofilm on the implant surface. Our group is developing a non-invasive method to eliminate biofilm on metal implants using alternating magnetic fields (AMF), utilizing the physical principle of electromagnetic induction. The aims of this dissertation are to establish exposure parameters for biofilm elimination in the presence and absence of antibiotics, the development of treatment strategies, and investigation the mechanism of AMF towards biofilm. First, the elimination of biofilm on metal implant using intermittent alternating magnetic field (iAMF) and antibiotics was studied, showing iAMF and antibiotics are synergistic in their biofilm reducing capability. For Pseudomonas aeruginosa biofilm, bacterial burden was reduced > 3 log with iAMF and ciprofloxacin after 24 h compared with either treatment alone. This additional treatment effect was also found on Staphylococcus aureus. iAMF and antibiotic efficacy was seen across various iAMF settings, including different iAMF target temperatures, dose durations, and dosing intervals. Initial mechanistic studies revealed membrane disruption as one factor important for AMF enhanced antibacterial activity in the biofilm. Then, the impact factors generated by AMF, heat and electric current, to metal implants were studied separately. A mathematic model was built to describe the response of biofilm to heat based on Arrhenius equation in order to study the responses of various strains to heat. Also, the synergistic effect of heat and antibiotics was observed towards biofilm elimination at various treatment temperatures. Finally, the effect of alternating electric current was studied using a burst AMF (bAMF) strategy, in which negligible heating was produced. For Pseudomonas aeruginosa biofilm, bAMF was able to eliminate biofilm in combination with antibiotics for surface current densities ranging from 99 to 297 A/cm2. This effect was also observed with bAMF and linezolid or rifampin in Staphylococcus aureus but with higher surface current densities required to achieve the same level of biofilm reduction. The results of this study support the use of AMF to reduce biofilm on infected metal implants. The effect can be achieved through both thermal and electrical pathways, or a combination of both. When combined with antibiotics, the effect is amplified and can regularly achieve biofilm eradication. In the future, the results of this thesis can be used to define operating parameters for the non-invasive treatment of infected metal implants.Item Polymorphonuclear Leukocyte Enhancement of Bacterial Biofilms on Contact Lens Surfaces(2015-01-26) Patel, Naiya; Hinojosa, Jorge A.; Zhu, Meifang; Robertson, Danielle M.Contact lens-wear represents a leading risk factor for the development of infectious keratitis, which can result in significant vision loss. It is well established that bacterial colonization of the posterior lens surface represents the initial event in the pathogenesis of lens-related infection. Using an invasive clinical isolate, our prior work has shown that Pseudomonas aeruginosa biofilm formation on contact lens surfaces is dramatically accelerated in the presence of dying neutrophils. The goal of this study was to investigate the capacity of five FDA test strains, all commonly associated with contact lens-related infiltrative events, to form biofilms on contact lens surfaces in the presence of neutrophil-derived cellular debris. Neutrophils were obtained from healthy, human volunteers by venipuncture and isolated using Ficoll gradient separation. Unworn Lotrafilcon B silicone hydrogel contact lenses were incubated overnight in one of five reference strains from the American Type Culture Collection with or without neutrophils at a 1:1 bacteria:neutrophil ratio. Test strains included: Pseudomonas aeruginosa, Staphylococcus epidermidis, Staphylococcus auerus, Stenotrophomonas maltophilia, and Serratia marcescens. Adherent bacteria were visualized using scanning electron microscopy (SEM) or stained using a BacLight live/dead assay followed by laser scanning confocal microscopy. The number of viable bacteria adherent to the lens surface was also quantified by standard colony counts. Live/dead staining showed greater numbers of viable bacteria adherent to lens surfaces when cultured in the presence of neutrophils. Colony counts confirmed a higher number of viable bacteria for four of the five test strains: S. auerus (p<0.001), S. maltophilia (p<0.001), P. aeruginosa (p=0.030), and S. marcescens (p<0.001). This effect was not evident with S. epidermidis (p=0.659). SEM showed similar findings. This is the first study to demonstrate the ability of these reference strains to form biofilms on contact lens surfaces in the presence of neutrophils. These findings suggest that, in the setting of intense inflammation under the lens, common contact lens-related pathogens possess the capacity to colonize and resist clearance by the innate immune system. Further studies are needed to correlate these findings with disease in an animal model.Item A Synthetic Quorum Sensing System Reveals Interaction Between Extracellular Matrix and Quorum Sensing Molecules(2015-09-29) Zhang, Fang; Ross, Elliott M.; Süel, Gürol M.; Sperandio, Vanessa; Liou, JenEven though bacteria are unicellular organisms, they commonly reside in structured communities known as biofilms. One of the defining characteristics of biofilms is the presence of an extracellular matrix (ECM) that encapsulates all cells within the community and provides the biofilm with structural integrity. The production and degradation of ECM components are often regulated by quorum sensing (QS), a prevailing cell-cell communication method between bacterial cells. Quorum sensing allows bacteria to communicate with each other by secreting and sensing small molecules called quorum signals. The literature suggests that the ECM may affect diffusion of quorum molecules through a physical connection between these processes. However, since QS regulates ECM expression, ECM expression and QS are tightly coupled and cannot be perturbed independently. Here we constructed a synthetic QS system in Bacillus subtilis to overcome this limitation and investigate whether ECM production affects QS signals, by quantitatively measuring the synthetic QS response in biofilm communities and single cells. Specifically, we constructed a synthetic quorum-sensing system with designated "Sender" and "Receiver" cells in Bacillus subtilis. This synthetic QS system allowed us to uncouple and independently investigate ECM production and QS in both biofilms and single cells. Our results showed that ECM-producing cells have a higher gene expression response to QS signals. The enhanced QS response suggests a private benefit for ECM-producing cells, which may indicate another mechanism to balance the cost of ECM production and constrain ECM production cheaters in biofilms.Item Thermal Eradication of Prosthetic Joint Associated Bacteria(2018-01-23) Kreutz, Kasey; Saini, Reshu; Chopra, Rajiv; Greenberg, DavidPURPOSE: As with other medical implants, prosthetic joints provide a hospitable surface for bacterial adherence and biofilm formation. We are developing a non-invasive thermal technique to destroy biofilm on the metal surfaces of prosthetic joints using alternating magnetic fields (AMF). One hypothesized benefit of AMF therapy is that it could be used in conjunction with traditional antibiotics to produce a synergistic bactericidal effect. The purpose of this study is to characterize the thermal sensitivity of bacteria in the presence of antibiotics to aid in the development of appropriate parameters for AMF dosing. METHODS: Planktonic solutions of Pseudomonas aeruginosa were thermally shocked in heating blocks with and without minimum inhibitory concentrations of ciprofloxacin. Bacterial solutions were plated on blood agar at intervals throughout the experiment to create a time-kill curve of the combined effects of heat shock therapy and ciprofloxacin. RESULTS: A single 10-minute dose of 55°C thermal shock reduced bacterial concentrations by 1.47-log CFU/mL, but subsequent incubation allowed bacteria to quickly reach and surpass original starting concentrations. Ciprofloxacin alone achieved a 3.78-log reduction within six hours and prevented significant regrowth during 24 hours of incubation. In combination, thermal shock and antibiotic achieved a 6.20-log reduction within 24 hours. After 24 hours allowing for regrowth, combination therapy produced superior bactericidal effect when compared to antibiotic or heat shock alone. When multiple doses of heat were applied in conjunction with antibiotic exposure, a step-down effect over time was observed as the log reduction achieved by each successive heat shock was either preserved or accentuated by the simultaneous inhibitory activity of the antibiotic. CONCLUSION: The results of this study demonstrate that the bactericidal effects of thermal shock are enhanced by the addition of antibiotics, and appear to show a promising synergistic benefit over either treatment being used alone. Future steps will focus on applying combination therapy to biofilm in an AMF coil in order to determine how dose requirements may differ.