Poloxamer-Mediated Control of Biofilms on Polycarbonate Surfaces

Background: Polycarbonate (PC) is a type of plastic widely used in the food industry as components of food processing equipment and resusable food/water storage containers and bottles. Biofilm formation on food contact surfaces (FCS) such as reusable plastics like PC can pose an potential health risk if insufficiently controlled. Poloxamers have surfactant properties that make them useful as dispersants, detergents, emulsifiers or wetting agents. Poloxamers have been utilised as an emulsifing agent and food additive in the food industry but their application in biofilm control on FCS has not been exploited to date. In particular, the soaking of PC items in poloxamers prior to routine washing processes may present a novel biofilm control measure for reusable products made from PC. Thus, this study investigated the potential use of poloxamers to control biofilm formation on nutrient-exposed PC surfaces. Methods: Poloxamer surfactants (P188 and P407: 0.1, 1.0 and 10.0 mg/ml) were investigated to assess their ability to disrupt biofilms of Gram-positive and Gram-negative bacteria. Staphylococcus aureus (Sa) & Pseudomonas aeruginosa (Pa) biofilms were cultured in nutrient-rich medium on PC surfaces (13mm PC discs; n=6) prior to exposure to poloxamer for 24 hours. The crystal violet (CV) assay was used to determine biofilm mass following treatment of Sa and Pa biofilms with poloxamers. The recovery of Sa or Pa from PC surfaces (n=6) following treatment of biofilms with poloxamer was assessed and biofilm disruption was visualised by confocal and scanning electron microscopy (SEM). Results: Both poloxamer P188 and P407 significantly (P<0.05) reduced Sa and Pa biofilm mass on nutrient-exposed PC surfaces in a dose-dependent manner. In concordance with this finding, treatment of established biofilms of Sa or Pa with poloxamer (P188 or P407) for 24 hours significantly (P<0.05) reduced bacterial recovery from nutrient-exposed PC surfaces. Confocal microscopy and SEM showed extensive biofilm disruption on poloxamer-treated PC surfaces compared to untreated control PC surfaces. Conclusions: This study showed poloxamer surfactants significantly disrupt established biofilms on PC surfaces. These findings suggest treatment of FCS with poloxamers prior to routine washing may be a novel biofilm control measure in the food industry for reusable items made from PC

Estrogen promotes macrophage-mediated clearance of biofilms in an in vitro model of an infected diabetic foot ulcer

Estrogen Promotes Macrophage-Mediated Clearance of Biofilms in an in vitro Model of an Infected Diabetic Foot Ulcer. Amina Belboul, Mohamed El-Mohtadi, Kathryn Whitehead and Jason Ashworth Centre for Bioscience, Manchester Metropolitan University, Manchester, United Kingdom. Amina Belboul ([email protected]) Jason Ashworth ([email protected]) Abstract Background: Diabetic foot ulcer (DFU) infections are common due to defective phagocytic activity of innate immune cells and/or one or more invasive bacterial species evading host responses. DFU infections are the main cause of diabetes-related hospitalisation and are a major cause of diabetes-related amputation, often persisting in protective biofilm arrangements. Alleviating biofilm growth is often one of the primary aims of DFU infection management. With antibiotic resistance now commonplace, there is an urgent need to reduce the reliance on antibiotics and develop novel treatments to combat wound infections. One novel strategy is to stimulate natural innate immune responses through increased phagocyte activity. The endogenous hormone estrogen is reported to be a master regulator of innate inflammatory responses, with declining levels of estrogen with increasing age leading to impaired wound healing. However, the effect of estrogen supplementation on innate host responses in DFU infections, particularly those involving biofilms, remains largely unknown. Thus, the aim of this study was to investigate the potential use of estrogen to promote macrophage-mediated clearance of two nosocomial wound-associated pathogens, methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (PA), under diabetic (hyperglycaemic) conditions using an in vitro biofilm model of an infected DFU. Materials/methods: In vitro host-pathogen interaction assays were used to determine the effect of estrogen (17-β estradiol) on the clearance of MRSA and PA biofilms (n=12) by U937-derived M1 macrophages cultured under increasingly hyperglycaemic (>11mM glucose) conditions. To confirm bacterial uptake and biofilm clearance by the estrogen-stimulated macrophages, the phagocytic activity of monocyte-derived macrophages was visualised by confocal and scanning electron microscopy (SEM). Results: Hyperglycaemia significantly (P<0.05; n=12) impaired M1 macrophage-mediated clearance of both MRSA and PA biofilms in a concentration-dependent manner, suggesting poorly controlled hyperglycaemia in diabetics may dampen phagocyte activity. Intriguingly, estrogen (1x10-7M) significantly (P<0.05; n=12) reversed the detrimental effects of hyperglycaemia on both MRSA and PA biofilm clearance. Pronounced interaction and uptake of bacteria by estrogen-treated M1 macrophages was visualised by confocal microscopy and SEM, confirming enhanced biofilm clearance compared with untreated controls. Conclusions: The findings suggest estrogen can reverse the detrimental effects of hyperglycaemia on M1 macrophage-mediated phagocytosis of key wound-associated bacteria, with estrogen enhancing the clearance of established biofilms. This highlights the possibility that topical estrogen application or localised estrogen receptor activation at the wound site might be effective therapeutic strategies to tackle the protective biofilm environments of DFU infections

Effect of Hormone-Driven Ageing on Inflammatory Cell Clearance of Bacteria under Hyperglycaemic Conditions

Background: Diabetic Foot Ulcers (DFUs) typically become colonised by a variety of bacteria, often persisting in protective biofilm arrangements. In most cases the bacterial colonisation is kept in check by host immunity but some DFUs can become infected due to defective phagocyte activity and/or one or more bacterial species evading host responses. With antibiotic resistance now commonplace, there is an urgent need to reduce the reliance on antibiotics and develop novel treatments to combat wound infections. One novel strategy is to stimulate natural innate immune responses through increased phagocyte activity. The endogenous hormone estrogen is reported to be a master regulator of innate inflammatory responses, with declining levels of estrogen with increasing age leading to impaired healing. However, the effect estrogen supplementation might have on innate host responses in infected DFUs remains largely unknown. Thus, the aim of this study was to investigate the potential use of estrogen to promote macrophage-mediated clearance of two problematic wound-associated pathogens, methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (PA), under diabetic (hyperglycaemic) conditions using a biofilm model of infected DFUs. Materials/methods: In vitro host-pathogen interaction assays were used to determine the effect of estrogen (17β-estradiol) on macrophage-mediated clearance of MRSA and PA biofilms (n=12) under increasing hyperglycaemic (>11mM glucose) conditions. The phagocytic activity of macrophages was visualised by fluorescence microscopy to confirm bacterial uptake. Results: Hyperglycaemia significantly (P<0.05; n=12) impaired macrophage-mediated clearance of both MRSA and PA biofilms in a concentration-dependent manner, suggesting poorly controlled hyperglycaemia in diabetics may dampen the phagocytic activity of macrophages. Intriguingly, estrogen significantly reversed the detrimental effects of hyperglycaemia on MRSA and PA biofilm clearance. Pronounced uptake of bacteria by estrogen-treated macrophages was visualised by fluorescence microscopy, confirming enhanced bacterial phagocytosis compared with untreated controls. Conclusions: The findings show estrogen reverses the detrimental effects of hyperglycaemia on macrophage-mediated phagocytosis of key wound-associated bacteria, even when such bacteria are established within a protective biofilm environment. This highlights the possibility that topical estrogen or localized estrogen receptor activation at the wound site might be effective therapeutic strategies to resolve infected DFUs

Estrogen augments the phagocytic function of macrophages through activation of estrogen-receptor alpha and actin cytoskeleton reorganisation

Background: Chronic wounds in the elderly often become infected, leading to substantial morbidity and mortality. Age-related impaired healing is mediated by age-related changes in steroid hormones, particularly declining levels of estrogen with increasing age. Although the anti-inflammatory activity of estrogen has been defined, little is known about the effects of estrogen deprivation on bacterial clearance. The aim of this study was to determine the effect of ageing (estrogen deprivation) on the ability of human monocyte-derived macrophages to eliminate bacteria via phagocytosis. Materials/methods: Host-pathogen assays were used to measure macrophage-mediated phagocytosis of two major wound pathogens, methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa, under in vitro and ex vivo conditions that model estrogen levels in the elderly, young adults and following estrogen supplementation. Fluorescence and scanning electron microscopy (SEM) were used to visualise host-pathogen interactions and protein mediators of phagocytosis were measured by immunoblotting. Results: Estrogen at concentrations typical of youth or supraphysiological levels significantly (P<0.05) increased the phagocytosis of MRSA and P. aeruginosa in a dose-dependent manner compared to estrogen deprivation with significantly enhanced clearance of bacteria by M1 macrophages compared to M2 macrophages. Epifluorescence, confocal and SEM confirmed estrogen increases co-localisation of fluorescent GFP-S. aureus or mCherry-P. aeruginosa within macrophages and promotes bacterial internalisation. Activation of estrogen receptor-alpha (ER-α) mirrored the stimulatory effect of estrogen on phagocytosis whilst ER-α antagonism significantly (P<0.01) blocked the phagocytic effect of estrogen. In contrast, activation of ER-beta (ER-β) had no significant effect on phagocytosis, confirming estrogen mediates bacterial clearance via ER-α. Immunoblotting analysis demonstrated that estrogen-enhanced phagocytosis is associated with altered levels of mediators involved in the actin cytoskeleton of phagocytes including increased levels of FAK, Rac1, Cdc42 and RhoG, but reduced levels of RhoA. Conclusions: Findings suggest estrogen may promote the resolution of wound infections during youth but this protection is lost as estrogen levels decline with increasing age, resulting in increased propensity and progression of age-related wound infections. Thus, novel wound dressings providing estrogen supplementation or selective activation of ER-α and/or specific targeting of downstream mediators of the actin cytoskeleton may provide effective treatment options for infected wounds in the elderly

Estrogen stimulates the clearance of wound bacteria by monocyte-derived macrophages via activation of estrogen-receptor alpha and actin cytoskeleton reorganization

Background: Chronic wounds in the elderly often become infected, leading to substantial morbidity and mortality. Age-related impaired healing is mediated by age-related changes in steroid hormones, particularly declining levels of estrogen with increasing age. Although the anti-inflammatory activity of estrogen has been defined, little is known about the effects of estrogen deprivation on bacterial clearance. The aim of this study was to determine the effect of ageing (estrogen deprivation) on the ability of human monocyte-derived macrophages to eliminate bacteria via phagocytosis. Materials/methods: Host-pathogen assays were used to measure macrophage-mediated phagocytosis of two major wound pathogens, methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa, under in vitro and ex vivo conditions that model estrogen levels in the elderly, young adults and following estrogen supplementation. Fluorescence and scanning electron microscopy (SEM) were used to visualise host-pathogen interactions and protein mediators of phagocytosis were measured by immunoblotting. Results: Estrogen at concentrations typical of youth or supraphysiological levels significantly (P<0.05) increased the phagocytosis of MRSA and P. aeruginosa in a dose-dependent manner compared to estrogen deprivation with significantly enhanced clearance of bacteria by M1 macrophages compared to M2 macrophages. Epifluorescence, confocal and SEM confirmed estrogen increases co-localisation of fluorescent GFP-S. aureus or mCherry-P. aeruginosa within macrophages and promotes bacterial internalisation. Activation of estrogen receptor-alpha (ER-α) mirrored the stimulatory effect of estrogen on phagocytosis whilst ER-α antagonism significantly (P<0.01) blocked the phagocytic effect of estrogen. In contrast, activation of ER-beta (ER-β) had no significant effect on phagocytosis, confirming estrogen mediates bacterial clearance via ER-α. Immunoblotting analysis demonstrated that estrogen-enhanced phagocytosis is associated with altered levels of mediators involved in the actin cytoskeleton of phagocytes including increased levels of FAK, Rac1, Cdc42 and RhoG, but reduced levels of RhoA. Conclusion: Findings suggest estrogen may promote the resolution of wound infections during youth but this protection is lost as estrogen levels decline with increasing age, resulting in increased propensity and progression of age-related wound infections. Thus, novel wound dressings providing estrogen supplementation or selective activation of ER-α and/or specific targeting of downstream mediators of the actin cytoskeleton may provide effective treatment options for infected wounds in the elderly

Differential engulfment of Staphylococcus aureus and Pseudomonas aeruginosa by monocyte-derived macrophages is associated with altered phagocyte biochemistry and morphology

Knowledge of changes in macrophages following bacterial engulfment is limited. U937-derived macrophages were incubated with Staphylococcus aureus or Pseudomonas aeruginosa. Morphological and biochemical changes in macrophages following host-pathogen interactions were visualized using Scanning Electron Microscopy (SEM) and Fourier-Transform Infrared Spectroscopy (FTIR) respectively. Principal Component Analysis (PCA) was used to assess the variability in the FTIR spectra. Following host-pathogen interactions, survival of S. aureus was significantly lower than P. aeruginosa (P 99 % of variability in the FTIR spectra explained by the first two principal components. These findings demonstrated that there were clear morphological and biochemical changes in macrophages following engulfment of two different bacterial types suggesting that the biochemical components of the bacterial cell wall influenced the biochemical characteristics and hence the morphology of macrophages in distinct ways