Enhanced Biofilm Eradication and Reduced Cytotoxicity of a Novel Polygalacturonic and Caprylic Acid Wound Ointment Compared with Common Antiseptic Ointments

BioMed Research International ◽

10.1155/2021/2710484 ◽

2021 ◽

Vol 2021 ◽

pp. 1-5

Author(s):

Bahgat Z. Gerges ◽

Joel Rosenblatt ◽

Y-Lan Truong ◽

Ruth A. Reitzel ◽

Ray Hachem ◽

...

Keyword(s):

Trypan Blue ◽

Caprylic Acid ◽

Methicillin Resistant Staphylococcus Aureus ◽

Multidrug Resistant ◽

Polygalacturonic Acid ◽

Alamar Blue ◽

Trypan Blue Exclusion ◽

Biofilm Model ◽

In Vivo Testing

Antiseptic wound ointments are widely used to treat dermal wounds that are microbially contaminated. Polygalacturonic acid (PG)+caprylic acid (CAP) is a novel combination that has been shown to eradicate biofilms. We developed a novel PG+CAP ointment and compared the biofilm eradication capability and cytotoxicity of PG+CAP with that of commercially available antiseptic wound ointments. We used a well-established biofilm model to quantitatively assess the eradication of organisms following exposure to the wound ointments for 2 hours. PG+CAP ointment completely eradicated Candida albicans, multidrug-resistant Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus biofilms, whereas MediHoney, polyhexamethylene biguanide (PHMB), and benzalkonium chloride (BZK) ointments failed to eradicate all biofilms within 2 hours. We assessed cytotoxicity by exposing L-929 fibroblasts to extracts of each ointment; Trypan blue exclusion was used to assess cell viability, and Alamar blue conversion was used to assess metabolic function. After exposure to PG+CAP and MediHoney, fibroblast viability was 96.23% and 95.23%, respectively (Trypan blue), and was comparable to untreated cells (98.77%). PHMB and BZK showed reduced viability (83.25% and 77.83%, respectively, p < 0.05 ). Metabolic activity results followed a similar pattern. Cytotoxicity of PG+CAP ointment towards erythrocytes was comparable to saline. PG+CAP ointment seems to be safe and can rapidly eradicate microbial biofilm; thus, PG+CAP ointment merits further in vivo testing as a potential antimicrobial wound ointment.

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683. Assessment of Biofilm Eradication and Cytotoxicity of a Novel Polygalacturonic Acid + Caprylic Acid Wound Ointment Compared with Antiseptic Wound Ointments

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz360.751 ◽

2019 ◽

Vol 6 (Supplement_2) ◽

pp. S311-S311

Author(s):

Bahgat Gerges ◽

Ruth A Reitzel ◽

Joel Rosenblatt ◽

Ray Y Hachem ◽

Issam I Raad

Keyword(s):

Metabolic Activity ◽

Caprylic Acid ◽

In Vivo Studies ◽

Polygalacturonic Acid ◽

Trypan Blue Exclusion ◽

Trypan Blue Exclusion Test ◽

Biofilm Model ◽

Ointment Base

Abstract Background Antiseptic wound ointments are increasing importance from safety, microbiological and public health points of view. Previously, Rosenblatt et al. (2017) has assessed polygalacturonic acid (PG) + caprylic acid (CAP) solution for biofilm eradication efficacy and cytotoxicity. In this study, we assessed biofilm eradication and cytotoxicity of PG+CAP wound ointment compared with commercially available wound ointment comparators. Methods Assessment of antimicrobial efficacy was conducted using a well-established biofilm model. Twenty-four-hour biofilm was formed on silicone discs and exposed wound ointments for 2 hours. Discs were then sonicated and cultured to quantitate any remaining viable biofilm. To assess cytotoxic effects of wound ointments, L-929 fibroblasts were exposed to 2% extracts of each ointment. The trypan exclusion test was used to access cell viability and Alamar blue was used to assess metabolic function. Ointments tested include, PG+CAP formulated in an inert ointment base, benzalkonium chloride quaternary ammonia antiseptic ointment (BZK), polyhexamethylene biguanide (PHMB) antiseptic ointment, and 2-hydroxyethylcellulose + glycerol inert ointment base. Untreated fibroblast cells were used as controls. Results Within 2 hours of exposure, PG+CAP ointment able to completely eradicate C. albicans (CA), MDR Pseudomonas aeruginosa (PS), and MRSA. Additionally, PG+CAP was significantly more efficacious than BZK for MRSA (P = 0.002) and PS (P = 0.015) and PHMB for MRSA (P = 0.02). In the trypan blue exclusion test PG+CAP yielded 96.29% viable cells compared with 77.83% and 83.25%, for the QUAT and PHMB ointments, respectively. Fibroblasts treated with 2% PG+CAP, retained 86.6% of metabolic activity compared with untreated cells while the QUAT and PHMB ointments retained 37.5% and 44.5% metabolic activity, respectively. Conclusion PG+CAP has enhanced effects on eradication of biofilm in vitro as well as less toxicity in vitro relative to the antiseptic wound ointments. Further in vivo studies are warranted. Disclosures All authors: No reported disclosures.

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Peroxidase-H2O2-halide system: Cytotoxic effect on mammalian tumor cells

Blood ◽

10.1182/blood.v45.2.161.bloodjournal452161 ◽

1975 ◽

Vol 45 (2) ◽

pp. 161-170 ◽

Cited By ~ 1

Author(s):

RA Clark ◽

SJ Klebanoff ◽

AB Einstein ◽

A Fefer

Keyword(s):

Thyroid Hormones ◽

Trypan Blue ◽

Cytotoxic Effect ◽

Enzyme System ◽

Inflammatory Cells ◽

51Cr Release ◽

Trypan Blue Exclusion ◽

System A ◽

Halide System

Myeloperoxidase, H2O2, and a halide constitute a potent antimicrobial system. A cytotoxic effect of this system on a line of mouse ascitic lymphoma cells (LSTRA) is demonstrated here using four different assay systems: 51Cr release, trypan blue exclusion, inhibition of glucose C-1 oxidation, and loss of oncogenicity for mice. Deletion of each component of the system, preheating the peroxidase, or addition of azide, cyanide, or catalase abolished the cytotoxicity. Myeloperoxidase was effective with either chloride or iodide as the halide, while lastoperoxidase was effective with iodide but not chloride. The iodinated thyroid hormones, triiodothyronine and thyroxine, could substitute for the halide, and H2O2 could be replaced by a peroxide- generating enzyme system such as glucose and glucose oxidase or by H2O2 producing bacteria such as pneumococci or streptococci. The possibility is raised that the peroxidases of inflammatory cells and certain biologic fluids may affect tumor initiation or growth in vivo.

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Lower threshold to NFκB activity sensitizes murine β-cells to streptozotocin

Journal of Endocrinology ◽

10.1530/joe-21-0047 ◽

2021 ◽

Author(s):

Clyde J. Wright ◽

Sarah McKenna ◽

Robyn De Dios ◽

Brit H. Boehmer ◽

Leanna Nguyen ◽

...

Keyword(s):

Cell Death ◽

Trypan Blue ◽

Target Genes ◽

Cell Injury ◽

Wild Type ◽

Β Cells ◽

Β Cell ◽

Trypan Blue Exclusion ◽

Min6 Cell

The β-cell response to injury may be as critical for the development of diabetes as the specific insult. In the current study, we use streptozotocin (STZ) to injure the β-cell in order to study the response with a focus on NFκB. MIN6 cells were exposed to STZ (0.5-8mM, 0-24h) ±TNFα (100ng/mL) and ±IκBβ siRNA to lower the threshold to NFκB activation. Cell viability was determined by trypan blue exclusion. NFκB activation was determined by expression of the target genes Nos2 and Cxcl10, localization of the NFκB proteins p65 and p50, and expression and localization of the NFκB inhibitors, IκBβ and IκBα. There was no NFκB activation in MIN6 cell exposed to STZ (2 mM) alone. However, knocking down IκBβ expression using siRNA resulted in STZ-induced expression of NFκB target genes and increased cell death, while co-incubation with STZ and TNFα enhanced cell death compared to either exposure alone. Adult male IκBβ-/- and wild type (WT) mice were exposed to STZ and monitored for diabetes. The IκBβ-/- mice developed hyperglycemia and diabetes more frequently than controls following STZ exposure. Based on these results we conclude that STZ exposure alone does not induce NFκB activity. However, lowering the threshold to NFκB activation by co-incubation with TNFα or lowering IκBβ levels by siRNA sensitizes the NFκB response to STZ and results in a higher likelihood of developing diabetes in vivo. Therefore, increasing the threshold to NFκB activation through stabilizing NFκB inhibitory proteins may prevent β-cell injury and the development of diabetes.

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1164. In vitro Antimicrobial Efficacy of Novel Antimicrobial Dacron Vascular Grafts in the Inhibition of Multidrug-Resistant Gram-Negative Biofilm

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz360.1027 ◽

2019 ◽

Vol 6 (Supplement_2) ◽

pp. S416-S416

Author(s):

Nylev Vargas-Cruz ◽

Joel Rosenblatt ◽

Ruth A Reitzel ◽

Kamal Khalil ◽

Issam I Raad

Keyword(s):

Biofilm Formation ◽

Bacterial Colonization ◽

Vascular Grafts ◽

Multidrug Resistant ◽

Antimicrobial Efficacy ◽

Gram Negative ◽

In Vivo Testing ◽

Colonization Model

Abstract Background Vascular graft infections can be a devastating complication in vascular reconstructive surgery. Management of these infections is highly invasive and includes excision of the graft, debridement of infected material, and in situ reconstruction. There is a need for additional strategies for infection prevention in graft surgeries. The minocycline + rifampin + chlorhexidine (MRCH) triple combination had been previously demonstrated to be effective against biofilm formation in central venous catheters. In this study, we evaluated in vitro effectiveness and durability of MRCH coated Dacron vascular grafts in inhibiting multidrug-resistant Gram-negative biofilm formation. Methods Dacron vascular grafts were coated with MRCH based on a proprietary method. Antimicrobial efficacy at baseline and 3-week durability was assessed using a well-established in vitro biofilm colonization model. Multidrug-resistant Gram-negative pathogens tested include CRE Escherichia coli (EC), MDR Pseudomonas aeruginosa (PS) and CRE Klebsiella pneumoniae (KB). Antimicrobial durability was assessed for grafts that had been eluting in serum for 3 weeks prior to testing. Baseline and 3-week grafts were quantitatively culture to enumerate any biofilm viable biofilm colonization. Uncoated Dacron grafts were used as controls. Results At baseline and 3 weeks MRCH vascular grafts completely inhibited biofilm formation resulting in an 8-log10 reduction in bacterial colonization compared with uncoated grafts. Conclusion MRCH coated Dacron vascular grafts demonstrated in vitro effectiveness for at least 3 weeks in preventing biofilm colonization by multidrug-resistant Gram-negative pathogens. Further in vivo testing is warranted. Disclosures All authors: No reported disclosures.

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Peroxidase-H2O2-halide system: Cytotoxic effect on mammalian tumor cells

Blood ◽

10.1182/blood.v45.2.161.161 ◽

1975 ◽

Vol 45 (2) ◽

pp. 161-170 ◽

Cited By ~ 102

Author(s):

RA Clark ◽

SJ Klebanoff ◽

AB Einstein ◽

A Fefer

Keyword(s):

Thyroid Hormones ◽

Trypan Blue ◽

Cytotoxic Effect ◽

Enzyme System ◽

Inflammatory Cells ◽

51Cr Release ◽

Trypan Blue Exclusion ◽

System A ◽

Halide System

Abstract Myeloperoxidase, H2O2, and a halide constitute a potent antimicrobial system. A cytotoxic effect of this system on a line of mouse ascitic lymphoma cells (LSTRA) is demonstrated here using four different assay systems: 51Cr release, trypan blue exclusion, inhibition of glucose C-1 oxidation, and loss of oncogenicity for mice. Deletion of each component of the system, preheating the peroxidase, or addition of azide, cyanide, or catalase abolished the cytotoxicity. Myeloperoxidase was effective with either chloride or iodide as the halide, while lastoperoxidase was effective with iodide but not chloride. The iodinated thyroid hormones, triiodothyronine and thyroxine, could substitute for the halide, and H2O2 could be replaced by a peroxide- generating enzyme system such as glucose and glucose oxidase or by H2O2 producing bacteria such as pneumococci or streptococci. The possibility is raised that the peroxidases of inflammatory cells and certain biologic fluids may affect tumor initiation or growth in vivo.

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L-Carnitine Does Not Reduce Cytotoxicity from Induction Chemotherapy for Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2018-99-117921 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5156-5156

Author(s):

Jessica Sea ◽

Etan Orgel ◽

Steven D. Mittelman

Keyword(s):

Fatty Acid ◽

Acute Lymphoblastic Leukemia ◽

Cell Viability ◽

Induction Chemotherapy ◽

Induction Therapy ◽

Trypan Blue ◽

Lymphoblastic Leukemia ◽

Amino Acid Derivative ◽

Alamar Blue ◽

Trypan Blue Exclusion

Abstract Introduction Off-target effects of induction therapy to treat Acute Lymphoblastic Leukemia (ALL) can lead to hepatotoxicity, cardiotoxicity, and neuropathy, particularly in patients with previous co-morbidities. Incorporation of pegylated L-asparaginase (L-ASP) into pediatric and adult ALL induction regimens has increased remission rate but results in severe hepatotoxicity. Levocarnitine (L-carnitine), a small amino-acid derivative, has been reported in small case series in children and adults with ALL to reduce asparaginase-induced hepatotoxicity. L-carnitine functions as a transport molecule and facilitates long-chain free fatty acid beta-oxidation; exogenous supplementation with L-carnitine may therefore protect hepatocytes through facilitation of fatty acid transport and maintenance of antioxidant balance in mitochondria. However, the effects of L-carnitine on ALL cell survival and possible contributions of L-carnitine to chemotherapy resistance in ALL are not known. Given the ability of L-carnitine to enhance energy production, we sought to investigate whether L-carnitine could compromise the efficacy of common induction chemotherapy agents against ALL cells. Methods Human ALL cells, BV173 and RS4;11 were treated with L-carnitine and either daunorubicin (DNR) or vincristine (VCR) for 72 hours (n=4-6). RS4;11 cells were additionally treated with L-ASP (n=4), and current testing to assess dexamethasone in RS4;11 is in progress. Experiments were performed in 96-well culture plates with a seeding density of 90,000-120,000 cells/well (~5 x105 cells/mL). Growth media (RPMI 1640, 1% sodium pyruvate, 1% glutamine, 10% fetal bovine serum) was supplemented to physiologic (50 μM) and supraphysiologic (100-200 μM) levels of L-carnitine (normal reference range: 25-70 μM). Chemotherapy doses were chosen to represent ~EC90 concentrations (BV173: 2 nM VCR and 25 nM DNR; RS4;11: 2 nM VCR, 20 nM DNR, and 0.1 IU/mL L-ASP). After 72 hours, cell viability was measured by trypan blue exclusion and confirmed with spectrophotometric analysis with the Alamar Blue cell viability assay. Data were analyzed using one-way analysis of variance (ANOVA) with significance defined as p<0.05. Results Addition of L-carnitine did not significantly affect cytotoxicity of VCR or DNR in BV173 (n=6) or cytotoxicity of VCR, DNR, or L-ASP in RS4;11 (n=4) as measured by trypan blue exclusion at 72 hours (Figure 1A). L-carnitine also did not affect cell viability assessed using Alamar Blue colorimetric assay, presented as the difference in reduction of resazurin (resorufin) compared to untreated RS4;11 or BV173 cells (Figure 1B). Conclusion Our data show L-carnitine has no significant effect on the cytotoxicity of VCR, L-ASP, or DNR in two human ALL cell lines. For providers incorporating L-carnitine into induction therapy to reduce asparaginase-induced hepatotoxicity, this data supports its use without compromising the efficacy of induction therapy. Further investigation is necessary to determine whether L-carnitine alters efficacy of glucocorticoid drugs, particularly dexamethasone, and other chemotherapies used in the treatment of ALL and across other ALL subtypes. Disclosures No relevant conflicts of interest to declare.

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