Browsing by Author "Kirker, Kelly R."

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Actin recruitment to the Chlamydia inclusion is spatiotemporally regulated by a mechanism that requires host and bacterial factors
(2012-10) Chin, E.; Kirker, Kelly R.; Zuck, Meghan; James, Garth A.; Hybiske, K.
The ability to exit host cells at the end of their developmental growth is a critical step for the intracellular bacterium Chlamydia. One exit strategy, extrusion, is mediated by host signaling pathways involved with actin polymerization. Here, we show that actin is recruited to the chlamydial inclusion as a late event, occurring after 20 hours post-infection (hpi) and only within a subpopulation of cells. This event increases significantly in prevalence and extent from 20 to 68 hpi, and actin coats strongly correlated with extrusions. In contrast to what has been reported for other intracellular pathogens, actin nucleation on Chlamydia inclusions did not â€˜flashâ€™, but rather exhibited moderate depolymerization dynamics. By using small molecule agents to selectively disrupt host signaling pathways involved with actin nucleation, modulate actin polymerization dynamics and also to disable the synthesis and secretion of chlamydial proteins, we further show that host and bacterial proteins are required for actin coat formation. Transient disruption of either host or bacterial signaling pathways resulted in rapid loss of coats in all infected cells and a reduction in extrusion formation. Inhibition of Chlamydia type III secretion also resulted in rapid loss of actin association on inclusions, thus implicating chlamydial effector proteins(s) as being central factors for engaging with host actin nucleating factors, such as formins. In conclusion, our data illuminate the host and bacterial driven process by which a dense actin matrix is dynamically nucleated and maintained on the Chlamydia inclusion. This late stage event is not ubiquitous for all infected cells in a population, and escalates in prevalence and extent throughout the developmental cycle of Chlamydia, culminating with their exit from the host cell by extrusion. The initiation of actin recruitment by Chlamydia appears to be novel, and may serve as an upstream determinant of the extrusion mechanism
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.
Differential effects of planktonic and biofilm MRSA on human fibroblasts
(2012-02) Kirker, Kelly R.; James, Garth A.; Fleckman, Philip; Olerud, John E.; Stewart, Philip S.
Bacteria colonizing chronic wounds often exist as biofilms, yet their role in chronic wound pathogenesis remains unclear. Staphylococcus aureus biofilms induce apoptosis in dermal keratinocytes, and given that chronic wound biofilms also colonize dermal tissue, it is important to investigate the effects of bacterial biofilms on dermal fibroblasts. The effects of a predominant wound pathogen, methicillin-resistant S.â€‰aureus, on normal, human, dermal fibroblasts were examined in vitro. Cell-culture medium was conditioned with equivalent numbers of either planktonic or biofilm methicillin-resistant S.â€‰aureus and then fed to fibroblast cultures. Fibroblast response was evaluated using scratch, viability, and apoptosis assays. The results suggested that fibroblasts experience the same fate when exposed to the soluble products of either planktonic or biofilm methicillin-resistant S.â€‰aureus, namely limited migration followed by death. Enzyme-linked immunosorbent assays demonstrated that fibroblast production of cytokines, growth factors, and proteases were differentially affected by planktonic and biofilm-conditioned medium. Planktonic-conditioned medium induced more interleukin-6, interleukin-8, vascular endothelial growth factor, transforming growth factor-Î²1, heparin-bound epidermal growth factor, matrix metalloproteinase-1, and metalloproteinase-3 production in fibroblasts than the biofilm-conditioned medium. Biofilm-conditioned medium induced more tumor necrosis factor-Î± production in fibroblasts compared with planktonic-conditioned medium, and suppressed metalloproteinase-3 production compared with controls.
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.
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, John
2-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.
In vitro studies evaluating the effects of biofilms on wound-healing cells: a review
(2017-04) Kirker, Kelly R.; James, Garth A.
Chronic wounds are characterized as wounds that have failed to proceed through the well-orchestrated healing process and have remained open for months to years. Open wounds are at risk for colonization by opportunistic pathogens. Bacteria that colonize the open wound bed form surface-attached, multicellular communities called biofilms, and chronic wound biofilms can contain a diverse microbiota. Investigators are just beginning to elucidate the role of biofilms in chronic wound pathogenesis, and have simplified the complex wound environment using in vitro models to obtain a fundamental understanding of the impact of biofilms on wound-healing cell types. The intent of this review is to describe current in vitro methodologies and their results. Investigations started with one host cell-type and single species biofilms and demonstrated that biofilms, or their secretions, had deleterious effects on wound-healing cells. More complex systems involved the use of multiple host cell/tissue types and single species biofilms. Using human skin-equivalent tissues, investigators demonstrated that a number of different species can grow on the tissue and elicit an inflammatory response from the tissue. A full understanding of how biofilms impact wound-healing cells and host tissues will have a profound effect on how chronic wounds are treated.
Loss of viability and induction of apoptosis in human keratinocytes exposed to Staphylococcus aureus biofilms in vitro
(2009-09) Kirker, Kelly R.; Secor, Patrick R.; James, Garth A.; Fleckman, Philip; Olerud, John E.; Stewart, Philip S.
Bacteria colonizing chronic wounds are believed to exist as polymicrobial, biofilm communities; however, there are few studies demonstrating the role of biofilms in chronic wound pathogenesis. This study establishes a novel method for studying the effect of biofilms on the cell types involved in wound healing. Cocultures of Staphylococcus aureus biofilms and human keratinocytes (HK) were created by initially growing S. aureus biofilms on tissue culture inserts then transferring the inserts to existing HK cultures. Biofilm-conditioned medium (BCM) was prepared by culturing the insert-supported biofilm in cell culture medium. As a control planktonic-conditioned medium (PCM) was also prepared. Biofilm, BCM, and PCM were used in migration, cell viability, and apoptosis assays. Changes in HK morphology were followed by brightfield and confocal microscopy. After only 3 hours exposure to BCM, but not PCM, HK formed dendritelike extensions and displayed reduced viability. After 9 hours, there was an increase in apoptosis (p 0.0004). At 24 hours, biofilm-, BCM-, and PCM-exposed HK all exhibited reduced scratch closure (p 0.0001). The results demonstrated that soluble products of both S. aureus planktonic cells and biofilms inhibit scratch closure. Furthermore, S. aureus biofilms significantly reduced HK viability and significantly increased HK apoptosis compared with planktonic S. aureus. Keratinocytes are the major cell type of the epidermis, which serves as the primary barrier between the external environment and the internal tissues. In this capacity, the epidermis also functions as a barricade to microorganisms, toxins, and various antigens. When the barrier is breached due to wounding, basal keratinocytes from the wound edges or dermal appendages migrate over the open wound to reestablish the barricade in a process called reepithelialization. Chronic wounds, such as diabetic foot ulcers, venous leg ulcers, and pressure ulcers, are characterized by prolonged inflammation, an altered wound matrix, and the failure to reepithelialize. Chronic wounds are also characterized as supporting a diverse microbial flora. A literature review by Bowler examined culture data from 62 published studies dating between 1969 and 1997.1 The most predominant wound isolate in both chronic and acute wounds was Staphylococcus aureus (reported in 63% of the studies), followed by coliforms (45%), Bacteroides spp. (39%), Peptostreptococcus spp. (36%), Pseudomonas aeruginosa (29%), Enterococcus spp. (26%), and Streptococcus pyogenes (13%).1 Using molecular techniques, Dowd et al.2 also demonstrated vast bacterial diversity within chronic wounds. The most prevalent species included Staphylococcus, Pseudomonas, Peptoniphilus, Enterobacter, Stenotrophomonas, Finegoldia, and Serratia spp. It has been speculated that bacteria colonizing chronic wounds exist as a biofilm.3–7 Biofilms represent bacterial communities surrounded by extracellular polysaccharide matrix. Such communities are often polymicrobial and resistant to antimicrobials. Chronic wounds are an ideal environment for bacterial infection and biofilm formation. The wound remains open for a prolonged period of time, increasing the odds of bacterial infection. The wound bed provides a surface for growth, and poor blood flow and hypoxia discourage native defenses.8 Studies have shown that wounds inoculated with bacteria form biofilms.6,9 Furthermore, in a recent study by James et al.,10 60% of chronic wound specimens were characterized as containing biofilm compared with 6% of acute wound specimens. Despite the prevalence of biofilms in wounds, there are few data illustrating the role of biofilms in chronic wound pathogenesis. This study establishes a novel method for directly studying the effect of biofilms on the cell types involved in wound healing. Specifically, it examines the effect of S. aureus biofilms on keratinocyte morphology, viability, and scratch closure. METHOD ANDMATERIALS Cell culture Normal human keratinocytes (HK) were isolated from newborn foreskin using methods previously described11 and in accordance with the University of Washington 690 Wound
Phevalin (aureusimine B) Production by Staphylococcus aureus Biofilm and Impacts on Human Keratinocyte Gene Expression
(2012-07) Secor, Patrick R.; Jennings, Laura K.; James, Garth A.; Kirker, Kelly R.; deLancey Pulcini, Elinor; McInnerney, Kathleen; Gerlach, Robin; Livinghouse, Tom; Hilmer, Jonathan K.; Bothner, Brian; Fleckman, Philip; Olerud, John E.; Stewart, Philip S.
Staphylococcus aureus biofilms are associated with chronic skin infections and are orders of magnitude more resistant to antimicrobials and host responses. S. aureus contains conserved nonribosomal peptide synthetases that produce the cyclic dipeptides tyrvalin and phevalin (aureusimine A and B, respectively). The biological function of these compounds has been speculated to be involved in virulence factor gene expression in S. aureus, protease inhibition in eukaryotic cells, and interspecies bacterial communication. However, the exact biological role of these compounds is unknown. Here, we report that S. aureus biofilms produce greater amounts of phevalin than their planktonic counterparts. Phevalin had no obvious impact on the extracellular metabolome of S. aureus as measured by high-performance liquid chromatography-mass spectrometry and nuclear magnetic resonance. When administered to human keratinocytes, phevalin had a modest effect on gene expression. However, conditioned medium from S. aureus spiked with phevalin amplified differences in keratinocyte gene expression compared to conditioned medium alone. Phevalin may be exploited as potential biomarker and/or therapeutic target for chronic, S. aureus biofilm-based infections.
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.
Quantitative NMR metabolite profiling of methicillin-resistant and methicillin-susceptible Staphylococcus aureus discriminates between biofilm and planktonic phenotypes
(2013-06) Ammons, Mary Cloud B.; Tripet, Brian P.; Carlson, Ross P.; Kirker, Kelly R.; Gross, M. A.; Stanisich, Jessica J.; Copie, Valerie
Wound bioburden in the form of colonizing biofilms is a major contributor to nonhealing wounds. Staphylococcus aureus is a Gram-positive, facultative anaerobe commonly found in chronic wounds; however, much remains unknown about the basic physiology of this opportunistic pathogen, especially with regard to the biofilm phenotype. Transcriptomic and proteomic analysis of S. aureus biofilms have suggested that S. aureus biofilms exhibit an altered metabolic state relative to the planktonic phenotype. Herein, comparisons of extracellular and intracellular metabolite profiles detected by 1H NMR were conducted for methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) S. aureus strains grown as biofilm and planktonic cultures. Principal component analysis distinguished the biofilm phenotype from the planktonic phenotype, and factor loadings analysis identified metabolites that contributed to the statistical separation of the biofilm from the planktonic phenotype, suggesting that key features distinguishing biofilm from planktonic growth include selective amino acid uptake, lipid catabolism, butanediol fermentation, and a shift in metabolism from energy production to assembly of cell-wall components and matrix deposition. These metabolite profiles provide a basis for the development of metabolite biomarkers that distinguish between biofilm and planktonic phenotypes in S. aureus and have the potential for improved diagnostic and therapeutic use in chronic wounds.
Streptococcus mutans and actinomyces naeslundii interaction in dual-species biofilm
(MDPI AG, 2020-01) de Oliveira, Rosa Virginia Dutra; Bonafé, Fernanda Salloume Sampaio; Spolidorio, Denise Madalena Palomari; Koga-Ito, Cristiane Yumi; de Farias, Aline Leite; Kirker, Kelly R.; James, Garth A.; Brighenti, Fernanda Lourenção
The study of bacterial interaction between Streptococcus mutans and Actinomyces naeslundii may disclose important features of biofilm interspecies relationships. The aim of this study was to characterize—with an emphasis on biofilm formation and composition and metabolic activity—single- and dual-species biofilms of S. mutans or A. naeslundii, and to use a drip flow reactor (DFR) to evaluate biofilm stress responses to 0.2% chlorhexidine diacetate (CHX). Single- and dual-species biofilms were grown for 24 h. The following factors were evaluated: cell viability, biomass and total proteins in the extracellular matrix, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide—“XTT”—reduction and lactic acid production. To evaluate stress response, biofilms were grown in DFR. Biofilms were treated with CHX or 0.9% sodium chloride (NaCl; control). Biofilms were plated for viability assessment. Confocal laser-scanning microscopy (CLSM) was also performed. Data analysis was carried out at 5% significance level. S. mutans viability and lactic acid production in dual-species biofilms were significantly reduced. S. mutans showed a higher resistance to CHX in dual-species biofilms. Total protein content, biomass and XTT reduction showed no significant di erences between singleand dual-species biofilms. CLSM images showed the formation of large clusters in dual-species biofilms. In conclusion, dual-species biofilms reduced S. mutans viability and lactic acid production and increased S. mutans’ resistance to chlorhexidine.
Testing wound dressings using an in vitro wound model
(2010-06) Lipp, C.; Kirker, Kelly R.; Agostinho, Alessandra; James, Garth A.; Stewart, Philip S.
Objective: To determine whether or not there are any significant differences in the effects of wound dressings on bacterial bioburden. Method: A selection of non-occlusive, non-adhesive dressings was tested for their effect on bacterial bioburden. The dressings selected included two dressings with antimicrobial properties (one containing silver and one containing PHMB), a cotton-based dressing enclosed in a perforated sleeve of poly(ethylene terephthalate), a carboxymethyl cellulose-based dressing, a fiber-free alginate dressing, and a 12-ply 100% cotton gauze. Using the colony-drip flow reactor (DFR) model, a meticillin-resistant Staphylococcus aureus (MRSA) or Pseudomonas aeruginosa biofilm was grown underneath a dressing sample. Biofilm growth was examined via plate counts, fluorescent microscopy and scanning electron microscopy. Results: The dressings containing antimicrobial agents had the greatest effect on bacterial load. In the MRSA experiments, both antimicrobial dressings produced lower bacteria counts than the other dressings (p<0.001), while in the P. aeruginosa experiments, only the silver-containing sample had fewer bacteria (p<0.0001). However, neither antimicrobial dressing was able to completely eradicate the bacteria when testing with either microorganism. Conclusion: The results presented herein illustrate that bacteria can grow unchallenged within the dressing environment and that an antimicrobial dressing can limit this bacterial growth. Declaration of interest: None.