Microbial Killing Research Articles

Functional role of iNOS-Rac2 interaction in neutrophil extracellular traps (NETs) induced cytotoxicity in sepsis

BackgroundRecent reports from this lab have demonstrated a higher incidence of NETs, nitrosative, as well as oxidative stress, and have a direct correlation with the severity of sepsis and organ damage. However, the mechanistic perspective of NETs induced organ damage has not been understood at the cellular and molecular level. Interaction of inducible nitric oxide synthase (iNOS) with Rac2 in regulating reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation and its implications in microbial killing has been reported. This study was, therefore, undertaken in neutrophils of sepsis patients to investigate the functional importance of iNOS-Rac2 interaction in ROS/ RNS, peroxynitrite generation, NETs generation, and NETs mediated cell death. MethodsThe study was conducted on 100 patients with sepsis and 50 healthy volunteers. Interaction between iNOS and Rac2 was performed using co-immunoprecipitation and co-immunolabeling assay. Free radicals involving ROS and RNS were evaluated using cytochrome c reduction assay. NETs formation was evaluated by fluorescence microscopy. The cytotoxic effect of NETs was assessed on lung carcinoma cell line (A549) using colorimetric Alamar blue assay. ResultsEnhanced interaction between iNOS and Rac2 was found in sepsis neutrophils in comparison with control. This was accompanied by an increased level of superoxide (O2.−), nitric oxide (NO), and peroxynitrite (ONOO−) which were decreased in the presence of NAC, DPI, and 1400 W, signifying the role of iNOS-Rac2 interaction. Enhanced NETs release from activated sepsis neutrophils were abrogated in the presence of DPI. NETs from sepsis neutrophils exert a cytotoxic effect on lung epithelial cells (A549) in a concentration-dependent manner. ConclusionOur findings exhibit the functional role of iNOS-Rac2 interaction in ROS/RNS, peroxynitrite generation, NETs generation, and NETs mediated cell death.

Extraction and characterization of lectin from Cicerarietinum black and Prunusdulcis nut raw, its anti-inflammatory, antibacterial activity against oral pathogens

Lectins are proteins essential for the biological recognition of cells a key factor for killing microbes. Since, legumes are good source, we extracted lectin from Cicerarietinum Black (CiarBL), Prunusdulcis nut raw (PruDuNRL), characterized for its key properties. The protein content of CiarBL was 13.13 ± 4.53, 25.45 ± 3.93, specific activity was 472.97, 243.80HAU/mg protein and for Prunusdulcis nut raw it was 04.60 ± 1.60, 17.84 ± 1.41and specific activity was 365.71, 252.52 HAU/mg protein. The total hemeagglutination activity of both lectin was found to be 512HAU. Blood group specificity analysis showed specificity to O+ and B+ for CiarBL, and for PruDuNRL O+, A+, B+ human erythrocytes both the lectin was specific to goat blood among the animal blood tested. The hemeagglutination inhibition assay showed specificity to glucose, maltose alone for CiarBL. While, PruDuNRL was glucose, sucrose, maltose, xylose specific. Both exhibited good antioxidant activity but erythrocyte membrane stability was higher with CiarBL when compared to PruDuNRL. CiarBL demonstrated promising stability to urea. While, PruDuNRL depicted sensitivity to pH, temperature, urea but both showed stability to salts such as FeCl3, Na2So4, CaCl2. SDS PAGE, authenticate it as low molecular weight i.e. 20, 29 KDa for CiarBL, for PruDuNRL 20 to 60KDa, 115KDa, the molecular weight was ranging from low to high. The antibacterial effect against gram positive oral pathogenic bacteria showed good result for CiarBL against Streptococcus oralis, Streptococcus pyogens at all concentration studied, moderate activity with Propionibacterium acnes, Staphylococcus aureus at 100 µg/ml. For PruDuNRL, the antibacterial activity was found to be decreased in the order of Streptococcus pyogenes < Streptococcus oralis < Propionibacterium acnes < Staphylococcus aureus. The possible mechanism involved was lectin interaction with teichoic and teichuronic acids, peptidoglycans, lipopolysaccharides on the surface of the bacteria. Lectin possessing good antioxidant, anti-inflammatory activity could hold better pharmacological applications. So, the result obtained from the present study paves way for further research in various avenues of clinical microbiology.

Design of Photosensitizing Agents for Targeted Antimicrobial Photodynamic Therapy.

Photodynamic inactivation of microorganisms has gained substantial attention due to its unique mode of action, in which pathogens are unable to generate resistance, and due to the fact that it can be applied in a minimally invasive manner. In photodynamic therapy (PDT), a non-toxic photosensitizer (PS) is activated by a specific wavelength of light and generates highly cytotoxic reactive oxygen species (ROS) such as superoxide (O2−, type-I mechanism) or singlet oxygen (1O2*, type-II mechanism). Although it offers many advantages over conventional treatment methods, ROS-mediated microbial killing is often faced with the issues of accessibility, poor selectivity and off-target damage. Thus, several strategies have been employed to develop target-specific antimicrobial PDT (aPDT). This includes conjugation of known PS building-blocks to either non-specific cationic moieties or target-specific antibiotics and antimicrobial peptides, or combining them with targeting nanomaterials. In this review, we summarise these general strategies and related challenges, and highlight recent developments in targeted aPDT.

Mitochondrial Ca2+ Signaling Is an Electrometabolic Switch to Fuel Phagosome Killing

SUMMARYPhagocytes reallocate metabolic resources to kill engulfed pathogens, but the intracellular signals that rapidly switch the immunometabolic program necessary to fuel microbial killing are not understood. We report that macrophages use a fast two-step Ca2+ relay to meet the bioenergetic demands of phagosomal killing. Upon detection of a fungal pathogen, macrophages rapidly elevate cytosolic Ca2+ (phase 1), and by concurrently activating the mitochondrial Ca2+ (mCa2+) uniporter (MCU), they trigger a rapid influx of Ca2+ into the mitochondria (phase 2). mCa2+ signaling reprograms mitochondrial metabolism, at least in part, through the activation of pyruvate dehydrogenase (PDH). Deprived of mCa2+ signaling, Mcu−/− macrophages are deficient in phagosomal reactive oxygen species (ROS) production and defective at killing fungi. Mice lacking MCU in their myeloid cells are highly susceptible to disseminated candidiasis. In essence, this study reveals an elegant design principle that MCU-dependent Ca2+ signaling is an electrometabolic switch to fuel phagosome killing.

Antioxidant Vitamins Are Correlated with Different Aspects of Phagocytic Processes in Healthy Nigerians: Benefits As Supplements During Antimicrobial Treatment

Background: Antioxidant vitamins are important for the immune system to function efficiently through several mechanisms. However, according to several previous studies, individual step of leucocyte phagocytosis is not correlated with different antioxidant vitamins.&#x0D; Methods: This study included 50 healthy Nigerians whose cellular phagocytic mechanism such as percentage leucocyte migration (%LM) and intracellular killing (%Nitroblue Tetrazolium Test) were determined by microscopy, neutrophil chemokines [plasma interleukin 8 (IL-8)] was determined using ELISA, and respiratory burst indices [plasma catalase (CAT), superoxide dismutase (SOD), myeloperoxidase (MPO), hydrogen peroxide (H2O2), and nitric oxide (NO)] were determined by spectrophotometry. While the plasma antioxidant vitamins (Vitamins A, C, and E) were determined using HPLC, the phagocytic indices, chemokines, and respiratory burst indices were correlated with plasma antioxidant vitamins using Spearman’s Correlation analysis at α0.05.&#x0D; Results: The results show that although among the healthy Nigerian adults, vitamin C was significantly and positively correlated with %NBT, it was negatively correlated with CAT activity. Vitamin A showed a significantly positive correlation with SOD while Vitamin E showed a significantly negative correlation with MPO.&#x0D; Conclusions: These findings suggest that antioxidant vitamins affect different stages of phagocytosis. It is advisable to use a combination of antioxidant vitamins as supplements with recommended treatment strategies against intracellular micro-organisms or inflammatory diseases.&#x0D; Keywords: Antioxidants, Intracellular microbial killing, Vitamins

COVID 19: The safety profile of common disinfectants used for sanitization

With the recent outbreak of COVID-19, sanitization is a must for the community as daily safety prevention. The common ingredients found in disinfectant including glycol, sodium hypochlorite, silver ion, iodophor iodine and polyvinyl-pyrrolidone iodine. An ideal disinfectant should have a wide anti-microbial spectrum, low dermatologically toxicity profile, and stable storage properties. Glycol is widely used as a personal care product due to its low toxicity profile. Although it has a wide anti-microbial spectrum, it must be co-formulated with other agent due to its lower microbial killing strength. It has been demonstrated that sodium hypochlorite is not only capable of destroying bacteria and viruses but also about inactivating viral antigens by breaking down the high concentration protein resulting in the isolation of necrotic tissue particles. Yet, due to its strong alkaline pH and oxidizing capacity, it can cause corrosive reaction when mucous membranes and skin are in touch with it. Besides, silver ion also plays an effective role in curbing SARS coronavirus. It is used widely in disinfecting medical equipment, wound therapy, and utilized in water purification systems by its bacteriostatic and active ingredient properties. Researches have proven that iodophor iodine is effective in against non-enveloped virus in which 1% of povidone-iodine is effective against coronaviruses. However, it can cause a chemical burn, blistering, and skin sloughing if it used as skin disinfection. This report will discuss the safety of these disinfectants in sanitization.

Antimicrobial Effectiveness of Calcium Silicate Sealers against a Nutrient-Stressed Multispecies Biofilm.

Purpose: This study compared the antimicrobial efficacy of calcium silicate sealers (BioRoot RCS and Total Fill BC) and conventional sealers (AH Plus and Tubli-seal) against planktonic bacteria and a nutrient-stressed multispecies biofilm. Methods: Antimicrobial properties of freshly mixed sealers were investigated using the direct contact test (DCT) and a nutrient-stressed multispecies biofilm comprised of five endodontic strains. Antimicrobial activity was determined using quantitative viable counts and confocal laser scanning microscopy (CLSM) analysis with live/dead staining. The pH of the sealers was analysed over a period of 28 days in Hanks Balanced Salt Solution (HBSS). Analysis of variance (ANOVA) with Tukey tests and the Kruskal–Wallis test were used for data analysis with a significance of 5%. Results: All endodontic sealers exhibited significant antimicrobial activity against planktonic bacteria (p < 0.05). BioRoot RCS caused a significant reduction in viable counts of the biofilms compared to AH Plus and the control (p < 0.05), while no significant difference could be observed compared to TotalFill BC and Tubli-seal (p > 0.05). CLSM analysis showed that BioRoot RCS and TotalFill BC exhibited significant biofilm inhibition compared to Tubli-seal, AH Plus and the control (p < 0.05). BioRoot RCS presented with the highest microbial killing, followed by TotalFill BC and Tubli-seal. Alkalizing activity was seen from the onset by BioRoot RCS, TotalFill BC and AH Plus. After 28 days, BioRoot RCS demonstrated the highest pH in HBSS (pH > 12). Conclusions: Calcium silicate sealers exhibited effective antimicrobial properties. This was demonstrated by superior biofilm inhibition capacity and microbial killing, with strong alkalizing activity compared to epoxy-based and zinc oxide-eugenol-based sealers.

Diagnostic Modalities Based on Flow Cytometry for Chronic Granulomatous Disease: A Multicenter Study in a Well-Defined Cohort

Chronic granulomatous disease (CGD) is characterized by defective microbial killing due to mutations affecting subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. Definitive genetic identification of disease subtypes may be delayed or not readily available. Sought to investigate the role of intracellular staining of NADPH oxidase enzyme subunits in predicting the respective genetic defects in patients with CGD and carriers. Thirty-four patients with genetically inherited CGD, including 12 patients with X-linked CGD (gp91phagocyte oxidase (phox) deficiency due to cytochrome b-245, beta polypeptide [CYBB] mutations) and 22 patients with autosomal-recessive CGD (p22phox, p47phox, and p67phox deficiency due to cytochrome b-245, alpha polypeptide [CYBA], neutrophil cytosolic factor 1 [NCF1] and NCF2 mutations, respectively) were recruited from different immunology centers and followed up prospectively. Dihydrorhodamine testing and NADPH oxidase subunit expression in white blood cells were determined by flow cytometry. gp91phox and p22phox defects, which result in simultaneous loss of both proteins due to their complex formation, were differentiated only by comparative analysis of patients' and mothers' intracellular staining. p47phox and p67phox protein expression was almost undetectable in patients compared with carrier mothers and healthy controls. The expression values of the respective subunits were found to be significantly higher in all controls as compared with carrier mothers, which in turn were higher than those of patients. Analysis of NADPH oxidase enzyme subunits by flow cytometry in patients and carriers is useful in the rapid prediction of the genetic defect of patients with CGD, thus guiding targeted sequencing and aiding in their early diagnosis.

Dalbavancin in the treatment of patients with Gram-positive diabetic foot infection: a case report

Dalbavancin is a potent lipoglycopeptide active against Gram+ bacteria with a favorable pharmacokinetic/pharmacodynamic (PK/PD) and safe­ty profile, suitable for numerous challenging infections. Dalbavancin has been approved for ABSSSIs, but its range of application and the relative posology is still debated. Diabetic foot infections are a major challenge and a leading source of complications, involving high direct and indirect costs for healthcare systems and for patients in terms of morbidity and disability. Gram+ bacteria are often causative agents in diabetic foot infections. We discuss here the case of a 43 yars old patient with complicated diabetic foot infection and the successful application of a dalbavancin secondary monotherapy treatment protocol. The patient, after suffering a two months complex hospitalisation gained wound haeling and limb rescue without any complication. We were also able to measures dalbavancin plasma lev­els determining its long half-life and high microbial killing ability. KEY WORDS dalbavancin, diabetic foot, gram-positive.

The Dichotomous Responses Driven by β-Defensins.

Defensins are short, rapidly evolving, cationic antimicrobial host defence peptides with a repertoire of functions, still incompletely realised, that extends beyond direct microbial killing. They are released or secreted at epithelial surfaces, and in some cases, from immune cells in response to infection and inflammation. Defensins have been described as endogenous alarmins, alerting the body to danger and responding to inflammatory signals by promoting both local innate and adaptive systemic immune responses. However, there is now increasing evidence that they exert variable control on the response to danger; creating a dichotomous response that can suppress inflammation in some circumstances but exacerbate the response to danger and damage in others and, at higher levels, lead to a cytotoxic effect. Focussing in this review on human β-defensins, we discuss the evidence for their functions as proinflammatory, immune activators amplifying the response to infection or damage signals and/or as mediators of resolution of damage, contributing to a return to homeostasis. Finally, we consider their involvement in the development of autoimmune diseases.

Developing Novel Host-Based Therapies Targeting Microbicidal Responses in Macrophages and Neutrophils to Combat Bacterial Antimicrobial Resistance.

Antimicrobial therapy has provided the main component of chemotherapy against bacterial pathogens. The effectiveness of this strategy has, however, been increasingly challenged by the emergence of antimicrobial resistance which now threatens the sustained utility of this approach. Humans and animals are constantly exposed to bacteria and have developed effective strategies to control pathogens involving innate and adaptive immune responses. Impaired pathogen handling by the innate immune system is a key determinant of susceptibility to bacterial infection. However, the essential components of this response, specifically those which are amenable to re-calibration to improve host defense, remain elusive despite extensive research. We provide a mini-review focusing on therapeutic targeting of microbicidal responses in macrophages and neutrophils to de-stress reliance on antimicrobial therapy. We highlight pre-clinical and clinical data pointing toward potential targets and therapies. We suggest that developing focused host-directed therapeutic strategies to enhance “pauci-inflammatory” microbial killing in myeloid phagocytes that maximizes pathogen clearance while minimizing the harmful consequences of the inflammatory response merits particular attention. We also suggest the importance of One Health approaches in developing host-based approaches through model development and comparative medicine in informing our understanding of how to deliver this strategy.

The Metabolic Basis of Immune Dysfunction Following Sepsis and Trauma.

Critically ill, severely injured and high-risk surgical patients are vulnerable to secondary infections during hospitalization and after hospital discharge. Studies show that the mitochondrial function and oxidative metabolism of monocytes and macrophages are impaired during sepsis. Alternatively, treatment with microbe-derived ligands, such as monophosphoryl lipid A (MPLA), peptidoglycan, or β-glucan, that interact with toll-like receptors and other pattern recognition receptors on leukocytes induces a state of innate immune memory that confers broad-spectrum resistance to infection with common hospital-acquired pathogens. Priming of macrophages with MPLA, CPG oligodeoxynucleotides (CpG ODN), or β-glucan induces a macrophage metabolic phenotype characterized by mitochondrial biogenesis and increased oxidative metabolism in parallel with increased glycolysis, cell size and granularity, augmented phagocytosis, heightened respiratory burst functions, and more effective killing of microbes. The mitochondrion is a bioenergetic organelle that not only contributes to energy supply, biosynthesis, and cellular redox functions but serves as a platform for regulating innate immunological functions such as production of reactive oxygen species (ROS) and regulatory intermediates. This review will define current knowledge of leukocyte metabolic dysfunction during and after sepsis and trauma. We will further discuss therapeutic strategies that target leukocyte mitochondrial function and might have value in preventing or reversing sepsis- and trauma-induced immune dysfunction.

Irgm1-deficiency leads to myeloid dysfunction in colon lamina propria and susceptibility to the intestinal pathogen Citrobacter rodentium.

IRGM and its mouse orthologue Irgm1 are dynamin-like proteins that regulate vesicular remodeling, intracellular microbial killing, and pathogen immunity. IRGM dysfunction is linked to inflammatory bowel disease (IBD), and while it is thought that defective intracellular killing of microbes underscores IBD susceptibility, studies have yet to address how IRGM/Irgm1 regulates immunity to microbes relevant to intestinal inflammation. Here we find that loss of Irgm1 confers marked susceptibility to Citrobacter rodentium, a noninvasive intestinal pathogen that models inflammatory responses to intestinal bacteria. Irgm1-deficient mice fail to control C. rodentium outgrowth in the intestine, leading to systemic pathogen spread and host mortality. Surprisingly, susceptibility due to loss of Irgm1 function was not linked to defective intracellular killing of C. rodentium or exaggerated inflammation, but was instead linked to failure to remodel specific colon lamina propria (C-LP) myeloid cells that expand in response to C. rodentium infection and are essential for C. rodentium immunity. Defective immune remodeling was most striking in C-LP monocytes, which were successfully recruited to the infected C-LP, but subsequently underwent apoptosis. Apoptotic susceptibility was induced by C. rodentium infection and was specific to this setting of pathogen infection, and was not apparent in other settings of intestinal inflammation. These studies reveal a novel role for Irgm1 in host defense and suggest that deficiencies in survival and remodeling of C-LP myeloid cells that control inflammatory intestinal bacteria may underpin IBD pathogenesis linked to IRGM dysfunction.

RANK Ligand Helps Immunity to Leishmania major by Skewing M2-Like Into M1 Macrophages.

Macrophages host Leishmania major infection, which causes cutaneous Leishmaniasis in humans. In the murine model, resistance to infection depends on the host immunity mediated by CD4 T-cell cytokines and macrophages. In association to other stimuli, the Th1 cytokine IFN-γ induces NO-mediated microbial killing by M1/classically-activated macrophages. By contrast, the Th2 cytokine IL-4 promotes M2/alternatively activated macrophages, which express arginase-1 and shelter infection. Other cytokines, such as RANKL, might also participate in the crosstalk between T cells and macrophages to restrict parasite infection. RANKL and its receptor RANK are known to play an essential role in bone remodeling, by inducing osteoclatogenesis. It has also been shown that RANKL stimulates antigen-presenting cells, such as DCs and macrophages, to enhance T cell responses. Here we investigated how RANKL directly modulates the effector macrophage phenotypes and immunity to L. major parasites. We found that inflammatory peritoneal macrophages from B6 mice express RANK and M2 features, such as CD301 (MGL) and CD206 (mannose receptor). Nonetheless, treatment with RANKL or IFN-γ induced macrophage differentiation into more mature F40/80hi macrophages able to produce IL-12 and TNF-α. In parallel, macrophages treated with RANKL, IFN-γ, or RANKL along with IFN-γ progressively downregulated the expression of the M2 hallmarks MGL, arginase-1, and CCL17. Moreover, a synergism between IFN-γ and RANKL enhanced inducible NO synthase (iNOS) expression and NO production by macrophages. These results are consistent with the idea that RANKL helps IFN-γ to induce a M2-like to M1 phenotype shift. Accordingly, concomitant treatment with RANKL and IFN-γ promoted macrophage-mediated immunity to L. major, by inducing NO and ROS-dependent parasite killing. Furthermore, by cooperating with IFN-γ, endogenous RANKL engages CD4 T-cell help toward L. major-infected macrophages to upregulate M1 and Th1 cytokine responses. Therefore, RANKL, in combination with IFN-γ, is a potential local therapeutic tool to improve immune responses in Leishmaniasis, by skewing M2-like into effector M1 macrophages.

The Orphan Immune Receptor LILRB3 Modulates Fc Receptor-Mediated Functions of Neutrophils.

Neutrophils are critical to the generation of effective immune responses and for killing invading microbes. Paired immune receptors provide important mechanisms to modulate neutrophil activation thresholds and effector functions. Expression of the leukocyte Ig-like receptor (LILR)A6 (ILT8/CD85b) and LILRB3 (ILT5/CD85a) paired-receptor system on human neutrophils has remained unclear because of the lack of specific molecular tools. Additionally, there is little known of their possible functions in neutrophil biology. The objective of this study was to characterize expression of LILRA6/LILRB3 receptors during human neutrophil differentiation and activation, and to assess their roles in modulating Fc receptor-mediated effector functions. LILRB3, but not LILRA6, was detected in human neutrophil lysates following immunoprecipitation by mass spectrometry. We demonstrate high LILRB3 expression on the surface of resting neutrophils and release from the surface following neutrophil activation. Surface expression was recapitulated in a human PLB-985 cell model of neutrophil-like differentiation. Continuous ligation of LILRB3 inhibited key IgA-mediated effector functions, including production of reactive oxygen species, phagocytic uptake, and microbial killing. This suggests that LILRB3 provides an important checkpoint to control human neutrophil activation and their antimicrobial effector functions during resting and early-activation stages of the neutrophil life cycle.

The effect of ozonation on dissolved oxygen and microbiological content in refill drinking water

One indicator of safe drinking water is its microbiological content of 0 colonies/100 ml. Ozonation is one method for killing microbes contained in drinking water. Disinfection in refillable drinking water that uses ozone needs to be studied, considering that the drinking water put into used gallons is washed again and can be drunk directly by consumers. It should be suspected that the drinking water used from drinking refill water contains bacteria, a dangerous virus. So the purpose of this study is to find out the microbiological content in refill drinking water with variable storage time for refill drinking water. The research method used is an observational research method. The parameters observed were temperature, pH, total microbes and dissolved oxygen content where the dissolved oxygen content indirectly indicates the reaction process of ozonation and bacterial activity contained in refill drinking water. The results showed that at pH showed a decrease from day to day, things this is caused by chemical reactions and microbiological activities. TPC shows that the smallest TPC value is 1 colony/100 ml on the second day, this is due to the ozone reaction in the water here H2O2 and (OH *) are very strong oxidizers which work optimally on the second day and then increase to 13 colonies/100 ml on day 5. The result of DO examination was an increase in DO on the second day to 8.2 mg/l which afterward finally decreased again the following day, this was due to an ozone reaction and bacterial activity in the water, therefore it was recommended that storage of drinking water after ozonation be stored for 2 days, and need renewal in the cleaning and disinfection of gallons to be used.

Construction of antimicrobial and biocompatible cotton textile based on quaternary ammonium salt from rosin acid

Antimicrobial cotton textiles (CT) show great promise for wound dressings. However, modifying CTs to have antimicrobial properties requires balancing the killing of microbes while protecting normal cells. In this study, the surface of CT was modified using maleopimaric acid quaternary ammonium cations (MPA-N+) from rosin acid. The surfaces morphology and chemical composition were determined by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS), which confirmed that the MPA-N+ modified CT (CT-g-MPA-N+) was prepared. CT-g-MPA-N+ shows strong and broad spectrum antimicrobial activities against Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa) and Gram-positive bacteria (Staphylococcus aureus). It also exhibits prominent durability of antimicrobial capability even after soaking in PBS for 6 days, and can effectively inhibit bacterial biofilm formation. Most importantly, the excellent biocompatibility of CT-g-MPA-N+ was verified by hemocompatible and cytotoxic assays. This work is believed to be promising method to prepare antimicrobial cotton textiles by surface modification and suggest the great potential application in wound dressing.

Ex vivo anti-microbial efficacy of various formaldehyde releasers against antibiotic resistant and antibiotic sensitive microorganisms involved in infectious keratitis

BackgroundCorneal infections with antibiotic-resistant microorganisms are an increasingly difficult management challenge and chemically or photochemically cross-linking the cornea for therapy presents a unique approach to managing such infections since both direct microbial pathogens killing and matrix stabilization can occur simultaneously. The present study was undertaken in order to compare the anti-microbial efficacy, in vitro, of 5 candidate cross-linking solutions against 5 different microbial pathogens with relevance to infectious keratitis.MethodsIn vitro bactericidal efficacy studies were carried out using 5 different FARs [diazolidinyl urea (DAU), 1,3-bis(hydroxymethyl)-5,5-dimethylimidazolidine-2,4-dione (DMDM), sodium hydroxymethylglycinate (SMG), 2-(hydroxymethyl)-2-nitro-1,3-propanediol (NT = nitrotriol), 2-nitro-1-propanol (NP)] against 5 different microbial pathogens including two antibiotic-resistant species [methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), Pseudomonas aeruginosa (PA), and Candida albicans (CA)]. Standard in vitro antimicrobial testing methods were used.ResultsThe results for MSSA were similar to those for MRSA. DAU, DMDM, and SMG all showed effectiveness with greater effects generally observed with longer incubation times and higher concentrations. Against MRSA, 40 mM SMG at 120 min showed a > 95% kill rate, p < 0.02. Against VRE, 40 mM DAU for 120 min showed a > 94% kill rate, p < 0.001. All FARs showed bactericidal effect against Pseudomonas aeruginosa, making PA the most susceptible of the strains tested. Candida showed relative resistance to these compounds, requiring high concentrations (100 mM) to achieve kill rates greater than 50%.ConclusionOur results show that each FAR compound has different effects against different cultures. Our antimicrobial armamentarium could potentially be broadened by DAU, DMDM, SMG and other FARs for antibiotic-resistant keratitis. Further testing in live animal models are indicated.

P021 The influence of short-chain fatty acids produced by commensal bacteria on macrophage phenotype

Abstract Background Macrophages are dynamic immune cells that react to environmental signals to adapt to their environment. Among this diverse array of signals are multiple bacterially derived metabolites including short-chain fatty acids (SCFAs) in the gastrointestinal tract. It has been previously shown that butyrate, one of several SCFAs produced by bacteria, is able to drive an antimicrobial phenotype in macrophages. However, it is still unknown which bacterial species are able to drive this impact and whether this effect can be recapitulated in vivo. The identification of a common molecular signature that drives this phenotype may allow us to easily identify bacteria with this beneficial phenotype and give insight into potential therapeutic strains. Methods Monocytes were differentiated into macrophages in the presence of SFCAs or bacterial supernatants from a wide range of gut commensals isolated from healthy human donors. RNA was isolated post-differentiation and gene expression screened using a defined gene panel using the Fluidgm Biomark system. In addition, cytokines present in the supernatant were measured by ELISA and the ability of the macrophages to kill intracellular bacteria determined. Currently, we are carrying out the same procedure using bone marrow-derived macrophages (BMDM) from mice in order to expand this work flow into the mouse system so that we can determine if bacteria identified can elicit their impact in vivo. Results We have screened numerous SCFAs alone and in combination in the bacterial killing assay to determine how these are able to influence macrophage phenotype. From these experiments, we have repeated the earlier observation that butyrate can increase macrophage killing of intracellular bacteria. In addition, we have identified four bacterial isolates that induce significantly increased microbial killing in macrophages. This is associated with the production of butyrate, acetate and formate. Using RNA from these different conditions, we are in the process of determining if there is a common molecular pathway by which this increased antimicrobial phenotype is driven. Conclusion Both SCFAs and SCFA producing bacteria are able to induce an antimicrobial phenotype in macrophages that increased the ability of these cells to kill intracellular pathogens. Identification of a common signature that drives these changes could help to screen bacterial isolates for this beneficial trait. Ongoing work hopes to identify such a signature, to enable us to screen large numbers of commensals using high-throughput molecular methods. This can then be verified using both in vitro and in vivo techniques.

Whole blood transcriptomic analysis of beef cattle at arrival identifies potential predictive molecules and mechanisms that indicate animals that naturally resist bovine respiratory disease.

Bovine respiratory disease (BRD) is a multifactorial disease complex and the leading infectious disease in post-weaned beef cattle. Clinical manifestations of BRD are recognized in beef calves within a high-risk setting, commonly associated with weaning, shipping, and novel feeding and housing environments. However, the understanding of complex host immune interactions and genomic mechanisms involved in BRD susceptibility remain elusive. Utilizing high-throughput RNA-sequencing, we contrasted the at-arrival blood transcriptomes of 6 beef cattle that ultimately developed BRD against 5 beef cattle that remained healthy within the same herd, differentiating BRD diagnosis from production metadata and treatment records. We identified 135 differentially expressed genes (DEGs) using the differential gene expression tools edgeR and DESeq2. Thirty-six of the DEGs shared between these two analysis platforms were prioritized for investigation of their relevance to infectious disease resistance using WebGestalt, STRING, and Reactome. Biological processes related to inflammatory response, immunological defense, lipoxin metabolism, and macrophage function were identified. Production of specialized pro-resolvin mediators (SPMs) and endogenous metabolism of angiotensinogen were increased in animals that resisted BRD. Protein-protein interaction modeling of gene products with significantly higher expression in cattle that naturally acquire BRD identified molecular processes involving microbial killing. Accordingly, identification of DEGs in whole blood at arrival revealed a clear distinction between calves that went on to develop BRD and those that resisted BRD. These results provide novel insight into host immune factors that are present at the time of arrival that confer protection from BRD.