Browsing by Author "Fisher, Steve T."

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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.
Biofilm penetration, triggered release and in vivo activity of inhaled liposomal amikacin in chronic Pseudomonas aeruginosa lung infections
(2008-02) Meers, P.; Neville, M.; Malinin, V.; Scotto, A. W.; Sardaryan, G.; Kurumunda, R.; Mackinson, C.; James, Garth A.; Fisher, Steve T.; Perkins, W. R.
Objectives: Chronic infections of Pseudomonas aeruginosa in the lungs of cystic fibrosis patients are intractable antibiotic targets because of their biofilm mode of growth. We have investigated the biofilm penetration, mechanism of drug release and in vivo antimicrobial activity of a unique nanoscale liposomal formulation of amikacin designed specifically for nebulization and inhaled delivery. Methods: Penetration of fluorescently labelled liposomes into sputum or P. aeruginosa (PA3064) biofilms was monitored by a filter assay and by epifluorescence or confocal scanning laser microscopy. Amikacin release in vitro and rat lung levels after inhalation of nebulized material were measured by fluorescence polarization immunoassay. A 14 day agar bead model of chronic Pseudomonas lung infection in rats was used to assess the efficacy of liposomal amikacin versus free aminoglycosides in the reduction of bacterial count. Results: Fluorescent liposomes penetrated readily into biofilms and infected mucus, whereas larger (1 µm) fluorescent beads did not. Amikacin release from liposomes was mediated by sputum or Pseudomonas biofilm supernatants. Rhamnolipids were implicated as the major releasing factors in these supernatants, active at one rhamnolipid per several hundred lipids within the liposomes. Inhaled liposomal amikacin was released in a slow, sustained manner in normal rat lungs and was orders of magnitude more efficacious than inhaled free amikacin in infected lungs. Conclusions: Penetration of biofilm and targeted, sustained release from liposomes can explain the superior in vivo efficacy of inhaled liposomal amikacin versus free drug observed in a 14 day infection model. Inhaled liposomal amikacin may represent an important therapy for chronic lung infections.
Detection of Pseudomonas aeruginosa biomarkers from thermally injured mice in situ using imaging mass spectrometry
(2017-12) Hamerly, Timothy; Everett, Jake A.; Paris, Nina; Fisher, Steve T.; Karunamurthy, Arivarasan; James, Garth A.; Rumbaugh, Kendra P.; Rhoads, Daniel D.; Bothner, Brian
Monitoring patients with burn wounds for infection is standard practice because failure to rapidly and specifically identify a pathogen can result in poor clinical outcomes, including death. Therefore, a method that facilitates detection and identification of pathogens in situ within minutes of biopsy would be a significant benefit to clinicians. Mass spectrometry is rapidly becoming a standard tool in clinical settings, capable of identifying specific pathogens from complex samples. Imaging mass spectrometry (IMS) expands the information content by enabling spatial resolution of biomarkers in tissue samples as in histology, without the need for specific stains/antibodies. Herein, a murine model of thermal injury was used to study infection of burn tissue by Pseudomonas aeruginosa. This is the first use of IMS to detect P. aeruginosa infection in situ from thermally injured tissue. Multiple molecular features could be spatially resolved to infected or uninfected tissue. This demonstrates the potential use of IMS in a clinical setting to aid doctors in identifying both presence and species of pathogens in tissue.
Efficacy of polyhexamethylene biguanide-containing antimicrobial foam dressing against MRSA relative to standard foam dressing
(2009-09) Kirker, Kelly R.; Fisher, Steve T.; James, Garth A.; McGhee, D.; Shah, Chirag B.
Many modern foam wound dressings possess a variety of attributes that are designed to create a supportive wound-healing environment. These attributes include absorbing exudate, providing optimum moisture balance at the wound surface, and preventing maceration of surrounding tissue. However, studies suggest that controlling wound bioburden should also be targeted when developing wound therapeutics. Thus, traditional foam dressings may absorb a copious amount of fluid, but may also provide an environment where microbes can grow unchallenged, leading to an increase in wound bioburden. However, antimicrobial foam dressings may prevent or reduce microbial growth, increasing the potential for wound healing. Studies reported herein evaluated the efficacy of 0.5% polyhexamethylene biguanide (PHMB) treated dressings to prevent the growth of methicillin- resistant Staphylococcus aureus (MRSA). An antimicrobial foam (Kendallâ„¢ AMD, Covidien, Mansfield, MA), which contains PHMB and a standard foam dressing (Copaâ„¢, Covidien, Mansfield, MA), which contains no PHMB (control), were directly inoculated with clinical isolate of MRSA and placed on a growth medium for selected time intervals. The presence or absence of microbial growth was quantified using plate counts and was visually assessed using scanning electron microscopy. At all time points, the antimicrobial foam dressing significantly reduced the MRSA growth compared to the control dressing. Similar results were also obtained in the microscopic evaluations.
In vitro susceptibility of established biofilms composed of a clinical wound isolate of Pseudomonas aeruginosa treated with lactoferrin and xylitol
(2009-03) Ammons, Mary Cloud B.; Ward, L. S.; Fisher, Steve T.; Wolcott, Randall D.; James, Garth A.
The medical impact of bacterial biofilms has increased with the recognition of biofilms as a major contributor to chronic wounds such as diabetic foot ulcers, venous leg ulcers and pressure ulcers. Traditional methods of treatment have proven ineffective, therefore this article presents in vitro evidence to support the use of novel antimicrobials in the treatment of Pseudomonas aeruginosa biofilm. An in vitro biofilm model with a clinical isolate of P. aeruginosa was subjected to treatment with either lactoferrin or xylitol alone or in combination. Combined lactoferrin and xylitol treatment disrupted the structure of the P. aeruginosa biofilm and resulted in a >2log reduction in viability. In situ analysis indicated that while xylitol treatment appeared to disrupt the biofilm structure, lactoferrin treatment resulted in a greater than two-fold increase in the number of permeabilised bacterial cells. The findings presented here indicated that combined treatment with lactoferrin and xylitol significantly decreases the viability of established P. aeruginosa biofilms in vitro and that the antimicrobial mechanism of this treatment includes both biofilm structural disruption and permeablisation of bacterial membranes.
Iron induces bimodal population development by Escherichia coli
(2013-01) DePas, W. H.; Hufnagel, D. A.; Lee, J. S.; Blanco, L. P.; Bernstein, Hans C.; Fisher, Steve T.; James, Garth A.; Stewart, Philip S.; Chapman, M. R.
Bacterial biofilm formation is a complex developmental process involving cellular differentiation and the formation of intricate 3D structures. Here we demonstrate that exposure to ferric chloride triggers rugose biofilm formation by the uropathogenic Escherichia coli strain UTI89 and by enteric bacteria Citrobacter koseri and Salmonella enterica serovar typhimurium. Two unique and separable cellular populations emerge in iron-triggered, rugose biofilms. Bacteria at the airâ€“biofilm interface express high levels of the biofilm regulator csgD, the cellulose activator adrA, and the curli subunit operon csgBAC. Bacteria in the interior of rugose biofilms express low levels of csgD and undetectable levels of matrix components curli and cellulose. Iron activation of rugose biofilms is linked to oxidative stress. Superoxide generation, either through addition of phenazine methosulfate or by deletion of sodA and sodB, stimulates rugose biofilm formation in the absence of high iron. Additionally, overexpression of Mn-superoxide dismutase, which can mitigate iron-derived reactive oxygen stress, decreases biofilm formation in a WT strain upon iron exposure. Not only does reactive oxygen stress promote rugose biofilm formation, but bacteria in the rugose biofilms display increased resistance to H2O2 toxicity. Altogether, we demonstrate that iron and superoxide stress trigger rugose biofilm formation in UTI89. Rugose biofilm development involves the elaboration of two distinct bacterial populations and increased resistance to oxidative stress.
Potency and penetration of telavancin in staphylococcal biofilms
(2015-10) Kirker, Kelly R.; Fisher, Steve T.; James, Garth A.
Due to the emergence of staphylococcal biofilm infections, the need for advanced antibiotics is crucial. The aim of this investigation was to evaluate the potency and penetration of telavancin against staphylococcal biofilms using two different biofilm models. Multiple staphylococcal strains, including meticillin-sensitive Staphylococcus aureus ATCC 29213, vancomycin-intermediate S. aureus ATCC 700787, heterogeneously vancomycin-intermediate S. aureus ATCC 700698 and meticillin-sensitive Staphylococcus epidermidis ATCC 12228, were grown and treated in drip-flow reactors to determine log reductions due to telavancin treatment. After 3 days of biofilm growth and 24 h of treatment, mean log reductions for telavancin ranged from 1.65 to 2.17 depending on the bacterial strain tested. Penetration was evaluated qualitatively using confocal scanning laser microscopy to image the infiltration of fluorescently labelled antibiotic into a staphylococcal biofilm grown in a flow cell. Fluorescently labelled telavancin rapidly penetrated the biofilms with no alteration in the biofilm structure.
Propionibacterium acnes biofilm is present in intervertebral discs of patients undergoing microdiscectomy
(2017-04) Capoor, Manu N.; Ruzicka, Filip; Schmitz, Jonathan E.; James, Garth A.; Machackova, Tana; Jancalek, Radim; Smrcka, Martin; Lipina, Radim; Ahmed, Fahad S.; Alamin, Todd F.; Anand, Neel; Baird, John C.; Bhatia, Nitin; Demir-Deviren, Sibel; Eastlack, Robert K.; Fisher, Steve T.; Garfin, Steven R.; Gogia, Jaspaul S.; Gokaslan, Ziya L.; Kuo, Calvin C.; Lee, Yu-Po; Mavrommatis, Konstantinos; Michu, Elleni; Noskova, Hana; Raz, Assaf; Sana, Jiri; Shamie, A. Nick; Stewart, Philip S.; Stonemetz, Jerry L.; Wang, Jeffrey C.; Witham, Timothy F.; Coscia, Michael F.; Birkenmaier, Christof; Fischetti, Vincent A.; Slaby, Ondrej
Background In previous studies, Propionibacterium acnes was cultured from intervertebral disc tissue of ~25% of patients undergoing microdiscectomy, suggesting a possible link between chronic bacterial infection and disc degeneration. However, given the prominence of P. acnes as a skin commensal, such analyses often struggled to exclude the alternate possibility that these organisms represent perioperative microbiologic contamination. This investigation seeks to validate P. acnes prevalence in resected disc cultures, while providing microscopic evidence of P. acnes biofilm in the intervertebral discs. Methods Specimens from 368 patients undergoing microdiscectomy for disc herniation were divided into several fragments, one being homogenized, subjected to quantitative anaerobic culture, and assessed for bacterial growth, and a second fragment frozen for additional analyses. Colonies were identified by MALDI-TOF mass spectrometry and P. acnes phylotyping was conducted by multiplex PCR. For a sub-set of specimens, bacteria localization within the disc was assessed by microscopy using confocal laser scanning and FISH. Results Bacteria were cultured from 162 discs (44%), including 119 cases (32.3%) with P. acnes. In 89 cases, P. acnes was cultured exclusively; in 30 cases, it was isolated in combination with other bacteria (primarily coagulase-negative Staphylococcus spp.) Among positive specimens, the median P. acnes bacterial burden was 350 CFU/g (12 - ~20,000 CFU/g). Thirty-eight P. acnes isolates were subjected to molecular sub-typing, identifying 4 of 6 defined phylogroups: IA1, IB, IC, and II. Eight culture-positive specimens were evaluated by fluorescence microscopy and revealed P. acnes in situ. Notably, these bacteria demonstrated a biofilm distribution within the disc matrix. P. acnes bacteria were more prevalent in males than females (39% vs. 23%, p = 0.0013). Conclusions This study confirms that P. acnes is prevalent in herniated disc tissue. Moreover, it provides the first visual evidence of P. acnes biofilms within such specimens, consistent with infection rather than microbiologic contamination.