Browsing by Author "Pulcini, Elinor D."

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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.
Asiatic acid and corosolic acid enhance the susceptibility of Pseudomonas aeruginosa biofilms to tobramycin
(2007-03) Garo, Eliane; Elridge, Gary R.; Goering, Matt G.; Pulcini, Elinor D.; Hamilton, Martin A.
Asiatic acid and corosolic acid are two natural products identified as biofilm inhibitors in a biofilm inhibition assay. We evaluated the activities of these two compounds on Pseudomonas aeruginosa biofilms grown in rotating disk reactors (RDRs) in combination with tobramycin and ciprofloxacin. To determine the ruggedness of our systems, the antibiotic susceptibilities of these biofilms were assessed with tobramycin and ciprofloxacin. The biofilm bacteria produced in the RDR were shown to display remarkable tolerance to 10 µg/ml of ciprofloxacin, thus mimicking the tolerance observed in recalcitrant bacterial infections. These studies further demonstrate that a nonmucoid strain of P. aeruginosa can form a biofilm that tolerates ciprofloxacin at clinically relevant concentrations. Neither asiatic acid nor corosolic acid reduced the viable cell density of P. aeruginosa biofilms. However, both compounds increased the susceptibility of biofilm bacteria to subsequent treatment with tobramycin, suggesting asiatic acid and corosolic acid to be compounds that potentiate the activity of antibiotics. A similar statistical interaction was observed between ciprofloxacin and subsequent treatment with tobramycin.
Bacterial biofilms: A review of current research
(2001) Pulcini, Elinor D.
Biofilms provide bacterial cells with a protective environment that allows for survival from antibiotics and host defense mechanisms. In order to understand how to control biofilms, it is important to understand the complexity of the biofilm system. This is in overview of four areas of current biofilm research: Biofilm resistance to antimicrobials and host defense mechanisms, the complexity of biofilm structure, the possible existence of a biofilm phenotype, and the ramifications of cell cell communication within the biofilm.
Bacterial biofilms: A review of current research
(2001) Pulcini, Elinor D.
Biofilms provide bacterial cells with a protective environment that allows for survival from antibiotics and host defense mechanisms. In order to understand how to control biofilms, it is important to understand the complexity of the biofilm system. This is in overview of four areas of current biofilm research: Biofilm resistance to antimicrobials and host defense mechanisms, the complexity of biofilm structure, the possible existence of a biofilm phenotype, and the ramifications of cell cell communication within the biofilm.
Biofilms in chronic wounds
(2008-01) James, Garth A.; Swogger, E.; Wolcott, Randall D.; Pulcini, Elinor D.; Secor, Patrick R.; Sestrich, Jennifer; Costerton, J. William; Stewart, Philip S.
Chronic wounds including diabetic foot ulcers, pressure ulcers, and venous leg ulcers are a worldwide health problem. It has been speculated that bacteria colonizing chronic wounds exist as highly persistent biofilm communities. This research examined chronic and acute wounds for biofilms and characterized microorganisms inhabiting these wounds. Chronic wound specimens were obtained from 77 subjects and acute wound specimens were obtained from 16 subjects. Culture data were collected using standard clinical techniques. Light and scanning electron microscopy techniques were used to analyze 50 of the chronic wound specimens and the 16 acute wound specimens. Molecular analyses were performed on the remaining 27 chronic wound specimens using denaturing gradient gel electrophoresis and sequence analysis. Of the 50 chronic wound specimens evaluated by microscopy, 30 were characterized as containing biofilm (60%), whereas only one of the 16 acute wound specimens was characterized as containing biofilm (6%). This was a statistically significant difference (p<0.001). Molecular analyses of chronic wound specimens revealed diverse polymicrobial communities and the presence of bacteria, including strictly anaerobic bacteria, not revealed by culture. Bacterial biofilm prevalence in specimens from chronic wounds relative to acute wounds observed in this study provides evidence that biofilms may be abundant in chronic wounds.
Biofilms: sensing and signaling
(2001-05) Pulcini, Elinor D.
Biofilms are a community of surface-attached microorganisms that can have far-reaching effects. Biofilms are costly to industry and affect human health in a variety of ways. Research is only now beginning to discern the complexities of biofilm formation.Pulcini, E.D., "Biofilms: Sensing and Signaling," J. California Dental Association, 29(5):3 (2001).
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 7Â·39 Â± 0Â·45, 10Â·22 Â± 0Â·22 and 7Â·13 Â± 0.Â·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.
An in vitro comparison of intraluminal biofilm bacteria transfer of three peripheral intravenous valved blood control catheters
(2013-06) Ryder, M.; James, Garth A.; Pulcini, Elinor D.; Parker, Albert E.
The insertion of peripheral intravenous catheters (PIVC) is the most common invasive procedure performed by nurses. The new generation of PIVCs developed to reduce blood exposure during insertion utilizes additional internal components within the catheter hub. These components increase the internal surface area that is thought to increase biofilm formation and subsequent transfer of bacteria into the bloodstream. This raises concern for increased risk of bloodstream infection. The purpose of the this study is to compare biofilm formation and bacterial transfer rate between 3 valved blood control PIVCs in a clinically simulated in vitro model.
Isolation of potentially pathogenic Escherichia coli O157:h7 from the Ganges River
(2007-02) Hamner, Steve; Broadaway, Susan C.; Mishra, Veer B.; Tripathi, Anshuman; Mishra, Rajesh K.; Pulcini, Elinor D.; Pyle, Barry H.; Ford, Tim E.
Escherichia coli serotype O157:H7 was detected among bacteria collected from the Ganges River. O157:H7 isolates tested positive for stx1, stx2, and eae gene sequences. Identification of potentially pathogenic isolates from extensively used source water indicates that O157:H7 may be a significant but as yet underacknowledged public health concern in India.
Microbial ecology of human skin and wounds
(2008) James, Garth A.; Swogger, E.; Pulcini, Elinor D.
Human skin is a complex organ that provides protection and regulates our interaction with the outside environment. The skin is composed of three layers, which include the epidermis, dermis, and hypodermis. Skin appendages include hair follicles, sebaceous glands, and sweat glands. These appendages are unevenly distributed on the skin. The stratum corneum is the outer protecitve layer of the epidermis and is composed of dead cells that are regularly shed from the surface. The outer layers of the epidermis are inhabited by microorganisms considered permanent skin residents as well as transient microorganisms that do not normally grow and multiply on the skin. The number and types of microorganisms inhabiting the skin are influenced by the skin conditions, including the density and activity of sebaceous and sweat glands. The secretions of these glands provide nutrients and selective conditions that influence the composition of the reisident microflora. This community is composed primarily of Gram-positive bacteria, including staphylococci, micrococci, and corynebacteria as well as lipophilic yeasts (Malassezia). This resident microflora is believed to help prevent skin colonization by pathogenic microorganisms. However, under certain conditions, skin disease can be caused by members of the resident flora. Skin infections are most often the result of injury to the skin. Cutaneous wounds enable access of microorganisms to normally sterile tissue and provide a much different niche for microbial growth than does intact skin. In the case of acute wounds, the healing process, including immune response, is capable of stemming invasion by microbes and repairing the wound. However, in some cases wounds become chronic and fail to heal within a reasonable time frame. Most often chronic wounds afflict the ill and elderly with underlying disorders (e.g., diabetes) or weakened immune systems. Large bacterial populations in wounds have been correlated with delayed healing, and control of microbial infection is recognized as an important aspect of wound care. However, the role of specific microorganisms in preventing would healing remains unclear. Similar types of microorganisms have been isolated from both acute and chronic wounds, although the latter tend to harbor more anaerobic bacteria. Growth of microorganisms as biofilms in wounds may also contribute to the delayed healing and poor response to treatment of chronic wounds. Overall, human skin and cutaneous wounds are complex ecosystems harboring diverse communities of microorganisms. A better understanding of these ecosystems may lead to improvements in human health.
Microsensor and transcriptomic signatures of oxygen depletion in biofilms associated with chronic wounds.
(2016-04) James, Garth A.; Zhao, Alice Ge; Usui, Marcia L.; Underwood, Robert A.; Nguyen, Hung; Beyenal, Haluk; Pulcini, Elinor D.; Hunt, Alessandra Agostinho; Bernstein, Hans C.; Fleckman, Philip; Olerud, John E.; Williamson, Kerry S.; Franklin, Michael J.; Stewart, Philip S.
Biofilms have been implicated in delayed wound healing, although the mechanisms by which biofilms impair wound healing are poorly understood. Many species of bacteria produce exotoxins and exoenzymes that may inhibit healing. In addition, oxygen consumption by biofilms and by the responding leukocytes, may impede wound healing by depleting the oxygen that is required for healing. In this study, oxygen microsensors to measure oxygen transects through in vitro cultured biofilms, biofilms formed in vivo within scabs from a diabetic (db/db) mouse wound model, and ex vivo human chronic wound specimens was used. The results showed that oxygen levels within mouse scabs had steep gradients that reached minima ranging from 17 to 72 mmHg on live mice and from 6.4 to 1.1 mmHg on euthanized mice. The oxygen gradients in the mouse scabs were similar to those observed for clinical isolates cultured in vitro and for human ex vivo specimens. To characterize the metabolic activities of the bacteria in the mouse scabs, transcriptomics analyses of Pseudomonas aeruginosa biofilms associated with the db/db mice wounds was performed. The results demonstrated that the bacteria expressed genes for metabolic activities associated with cell growth. Interestingly, the transcriptome results also indicated that the bacteria within the wounds experienced oxygen-limitation stress. Among the bacterial genes that were expressed in vivo were genes associated with the Anr-mediated hypoxia-stress response. Other bacterial stress response genes highly expressed in vivo were genes associated with stationary-phase growth, osmotic stress, and RpoH-mediated heat shock stress. Overall, the results supported the hypothesis that bacterial biofilms in chronic wounds promote chronicity by contributing to the maintenance of localized low oxygen tensions, through their metabolic activities and through their recruitment of cells that consume oxygen for host defensive processes.
Natural green coating inhibits adhesion of clinically important bacteria
(2015-02) Trentin, D. S.; Silva, D. B.; Frasson, A. P.; Rzhepishevska, O.; da Silva, M. V.; Pulcini, Elinor D.; James, Garth A.; Soares, G. V.; Tasca, T.; Ramstedt, M.; Giordani, R. B.; Lopes, N. P.; Macedo, A. J.
Despite many advances, biomaterial-associated infections continue to be a major clinical problem. In order to minimize bacterial adhesion, material surface modifications are currently being investigated and natural products possess large potential for the design of innovative surface coatings. We report the bioguided phytochemical investigation of Pityrocarpa moniliformis and the characterization of tannins by mass spectrometry. It was demonstrated that B-type linked proanthocyanidins-coated surfaces, here termed Green coatings, reduced Gram-positive bacterial adhesion and supported mammalian cell spreading. The proposed mechanism of bacterial attachment inhibition is based on electrostatic repulsion, high hydrophilicity and the steric hindrance provided by the coating that blocks bacterium-substratum interactions. This work shows the applicability of a prototype Green-coated surface that aims to promote necessary mammalian tissue compatibility, while reducing bacterial colonization.
Production of cell–cell signaling molecules by bacteria isolated from human chronic wounds
(2010-05) Rickard, A. H.; Colacino, K. R.; Manton, K. M.; Morton, R. I.; Pulcini, Elinor D.; Pfeil, J.; Rhoads, Daniel D.; Wolcott, Randall D.; James, Garth A.
Aim: To (i) identify chronic wound bacteria and to test their ability to produce acyl-homoserine-lactones (AHLs) and autoinducer-2 (AI-2) cellâ€“cell signalling molecules and (ii) determine whether chronic wound debridement samples might contain these molecules. Methods and Results: Partial 16S rRNA gene sequencing revealed the identity of 46 chronic wound strains belonging to nine genera. Using bio-reporter assays, 69-6% of the chronic wound strains were inferred to produce AI-2, while 19-6% were inferred to produce AHL molecules. At least one strain from every genus, except those belonging to the genera Acinetobacter and Pseudomonas, were indicated to produce AI-2. Production of AI-2 in batch cultures was growth-phase dependent. Cross-feeding assays demonstrated that AHLs were produced by Acinetobacter spp., Pseudomonas aeruginosa and Serratia marcescens. Independent from studies of the bacterial species isolated from wounds, AHL and/or AI-2 signalling molecules were detected in 21 of 30 debridement samples of unknown microbial composition. Conclusion: Chronic wound bacteria produce cellâ€“cell signalling molecules. Based on our findings, we hypothesize that resident species generally produce AI-2 molecules, and aggressive transient species associated with chronic wounds typically produce AHLs. Both these classes of cellâ€“cell signals are indicated to be present in human chronic wounds. Significance and Impact of the Study: Interbacterial cellâ€“cell signalling may be an important factor influencing wound development and if this is the case, the presence of AHLs and AI-2 could be used as a predictor of wound severity. Manipulation of cellâ€“cell signalling may provide a novel strategy for improving wound healing.
Resident bacterial flora in the skin of C57BL/6 mice housed under SPF conditions
(2010-09) Tavakkol, Zarry; Samuelson, Derrick; Pulcini, Elinor D.; Underwood, Robert A.; Usui, Marcia L.; Costerton, J. William; James, Garth A.; Olerud, John E.; Fleckman, Philip
Research in cutaneous biology frequently involves models that use mice housed in SPF conditions. Little information is available concerning the species of bacteria that normally inhabit the skin of these mice. The aim of this study was to characterize the bacterial skin flora of mice housed under SPF conditions. Skin biopsies from C57BL/6 mice under normal and surgically prepped conditions were both cultured and analyzed by using DNA extraction and sequencing. The species isolated most commonly from culture were staphylococci. Coagulase-negative staphylococci were isolated more frequently than was Staphylococcus aureus. Molecular sequencing yielded several additional organisms not found by culture. Overall, culturing of isolates yielded 14 species of bacteria, and molecular sequencing identified another 6 species. Investigators conducting cutaneous research in mouse models should aware of the cutaneous bacterial flora present on these mice.
Surface-attached cells, biofilms and biocide susceptibility: Implications for hospital cleaning and disinfection
(2015-01) Otter, J. A.; Vickery, Karen; Walker, Johann; Pulcini, Elinor D.; Goldenberg, S. D.; Salkeld, J. A. G.; Chewins, J.; Yezli, S.; Edgeworth, J. D.
Microbes tend to attach to available surfaces and readily form biofilms, which is problematic in healthcare settings. Biofilms are traditionally associated with wet or damp surfaces such as indwelling medical devices and tubing on medical equipment. However, microbes can survive for extended periods in a desiccated state on dry hospital surfaces, and biofilms have recently been discovered on dry hospital surfaces. Microbes attached to surfaces and in biofilms are less susceptible to biocides, antibiotics and physical stress. Thus, surface attachment and/or biofilm formation may explain how vegetative bacteria can survive on surfaces for weeks to months (or more), interfere with attempts to recover microbes through environmental sampling, and provide a mixed bacterial population for the horizontal transfer of resistance genes. The capacity of existing detergent formulations and disinfectants to disrupt biofilms may have an important and previously unrecognized role in determining their effectiveness in the field, which should be reflected in testing standards. There is a need for further research to elucidate the nature and physiology of microbes on dry hospital surfaces, specifically the prevalence and composition of biofilms. This will inform new approaches to hospital cleaning and disinfection, including novel surfaces that reduce microbial attachment and improve microbial detachment, and methods to augment the activity of biocides against surface-attached microbes such as bacteriophages and antimicrobial peptides. Future strategies to address environmental contamination on hospital surfaces should consider the presence of microbes attached to surfaces, including biofilms.