Gilly Regev-Shoshani

Slow Release of Nitric Oxide from Charged Catheters and Its Effect on Biofilm Formation by Escherichia coli

ABSTRACT Catheter-associated urinary tract infection is the most prevalent cause of nosocomial infections. Bacteria associated with biofilm formation play a key role in the morbidity and pathogenesis of these infections. Nitric oxide (NO) is a naturally produced free radical with proven bactericidal effect. In this study, Foley urinary catheters were impregnated with gaseous NO. The catheters demonstrated slow release of nitric oxide over a 14-day period. The charged catheters were rendered antiseptic, and as such, were able to prevent bacterial colonization and biofilm formation on their luminal and exterior surfaces. In addition, we observed that NO-impregnated catheters were able to inhibit the growth of Escherichia coli within the surrounding media, demonstrating the ability to eradicate a bacterial concentration of up to 104 CFU/ml.

Comparative efficacy of commercially available and emerging antimicrobial urinary catheters against bacteriuria caused by E. coli in vitro.

OBJECTIVES To compare the efficacy of both commercially available and emerging urinary catheter technologies in relation to their effects on bacteriuria caused by Escherichia coli in vitro. Antiseptic urinary catheters have recently become commercially available and others are in the developmental stage. METHODS Silver alloy-coated catheters, antibiotic Nitrofurazone (NF)-coated catheters, and nitric oxide (NO)-coated catheters were tested against a noncoated control for their antiseptic ability. Inhibition of bacterial growth, biofilm formation, and the number of live bacteria within the biofilm, using up to 10(3) bacterial load were evaluated. Experiments were performed either in E. coli containing Luria broth media or in urine infected with E. coli. RESULTS NF- and NO-coated catheters had equivalent antimicrobial activity and eradicated all bacteria in planktonic and biofilm states. Silver-coated catheters had no effect on E. coli growth or biofilm formation compared with the control, although silver-coated catheters did inhibit bacterial levels within the biofilm by 50%. CONCLUSIONS NF- and NO-coated catheters are highly effective in preventing planktonic growth and biofilm formation. Silver-coated catheters were not found to be effective in this study.

A nitric oxide-releasing solution as a potential treatment for fungi associated with tinea pedis.

To test a nitric oxide‐releasing solution (NORS) as a potential antifungal footbath therapy against Trichophyton mentagrophytes and Trichophyton rubrum during the mycelial and conidial phases.

Gaseous nitric oxide reduces influenza infectivity in vitro

Abstract Gaseous nitric oxide (gNO) is an approved vasodilator drug for inhalation up to a maximum dose of 80ppm. While gNO has been shown, in vitro, to be an effective antibacterial agent (at 160ppm), NO-donor compounds have been shown to inhibit a variety of viruses at varying stages of replication. This research was done in order to determine whether gNO at 80 or 160ppm possesses an antiviral effect on influenza viruses. Three strains of influenza (A and B) were exposed to gNO for up to 180min, before and after infection of MDCK cells. In search for possible mechanism of antiviral action, Neuraminidase (NA) inhibition assay of H1N1 that was exposed to gNO was performed. Results show that when virions were exposed to gNO prior to infection a complete inhibition of infectivity was achieved for all three strains. Post infection exposure of influenza with gNO resulted in about 30% inhibition of infectivity. Further testing showed that when eliminating the pH effect by exposing a dried virus to gNO, 90% inhibition was found after 2h exposure. NA activity, of whole dried H1N1 virus, was found to be inhibited by gNO (80%). These results suggest that 80 and 160ppm gNO have a time dependent antiviral effect on influenza strains of viruses during various stages of cellular infection, which are not due to concomitant changes in pH in the surrounding milieu. Viral NA inhibition by gNO was shown and may be responsible for this antiviral effect.

Prophylactic nitric oxide treatment reduces incidence of bovine respiratory disease complex in beef cattle arriving at a feedlot

Bovine respiratory disease complex (BRDc), is a challenging multi-factorial health issue caused by viral/bacterial pathogens and stressors linked with the transport and mixing of cattle, negatively impacting the cattle feedlot industry. Nitric oxide (NO) is a naturally occurring molecule with antimicrobial attributes. This study tests whether NO can prevent the symptoms associated with BRDc. Eighty-five, crossbred, multiple-sourced, commingled commercial weaned beef calves were monitored and scored for temperature, white blood count, clinical score, hematology, cortisol levels and neutrophil/lymphocyte ratio. NO treatment or placebo were given once on arrival to the stockyard. After one week 87.5% of sick animals were from the control while 12.5% from treatment groups and after two weeks 72% and 28% respectively. Treatment was shown to be safe, causing neither distress nor adverse effects on the animals. These data show that NO treatment on arrival to the feedlot significantly decreased the incidence of BRDc in this study.

Safety, bioavailability and mechanism of action of nitric oxide to control Bovine Respiratory Disease Complex in calves entering a feedlot.

Bovine Respiratory Disease Complex (BRDc), a multi-factorial disease, negatively impacts the cattle industry. Nitric oxide (NO), a naturally occurring molecule, may have utility controlling incidence of BRDc. Safety, bioavailability, toxicology and tolerance/stress of administering NO to cattle is evaluated herein. Thirteen, crossbred, multiple-sourced, commingled commercial weaned beef calves were treated multiple times intranasally over a 4 week period with either a nitric oxide releasing solution (treatment) or saline (control). Exhaled NO, methemoglobin percent (MetHg) and serum nitrites demonstrated biological availability as a result of treatment. Cortisol levels, tissue nitrites, behavior and gross and macroscopic pathology of organs were all normal. Moreover, preliminary in vitro studies using Mannheimia haemolytica, Infectious Bovine Rhinotracheitis, Bovine Parainfluenza-3 and Bovine Respiratory Syncytial Virus, suggest a potential explanation for the previously demonstrated efficacy for BRDc. These data confirm the bioavailability, safety and lack of residual of NO treatment to cattle, along with the bactericidal and virucidal effects.

Effects of nasal instillation of a nitric oxide-releasing solution or parenteral administration of tilmicosin on the nasopharyngeal microbiota of beef feedlot cattle at high-risk of developing respiratory tract disease

Nitric oxide has bactericidal and virucidal properties. Nasal instillation of a nitric oxide releasing solution (NORS) on arrival at the feedlot was recently reported as inferior to a parenteral injection of tilmicosin (macrolide antibiotic) for control of bovine respiratory disease (BRD) in cattle at high-risk of developing BRD. We hypothesized that this inferiority was due to differences between treatments with regards to their effects on the nasopharyngeal microbiota. The objective was to compare nasal instillation of NORS versus parenteral administration of tilmicosin regarding their effects on the nasopharyngeal microbiota of feedlot cattle at high-risk of developing BRD. Culture-independent community profiling (16S rRNA sequencing) and culture-based methods were used to evaluate treatment effects. High-risk Angus-cross heifers (n=20) were randomly allocated to 2 treatment groups on arrival at a feedlot and received either NORS or tilmicosin for prevention of BRD. Heifers were sampled using guarded deep nasal swabs immediately prior to treatment (day 0) and on days 1, 5 and 10 after treatment. Based on culture-independent community profiling, there was a distinct shift in composition of the nasopharyngeal microbiota during the first 10 d after arrival, with 116 OTUs changing over time, but no difference between treatment groups. However, culture-based methods detected a difference between treatment groups, with more cattle culture-positive for Pasteurellaceae in the NORS group at day 5 post-treatment. This difference in ability to inhibit colonization of the nasopharynx by Pasteurellaceae may be the basis for NORS being inferior to tilmicosin for control of BRD in high-risk cattle.

Non-inferiority of nitric oxide releasing intranasal spray compared to sub-therapeutic antibiotics to reduce incidence of undifferentiated fever and bovine respiratory disease complex in low to moderate risk beef cattle arriving at a commercial feedlot

Undifferentiated fever, or bovine respiratory disease complex (BRDc), is a challenging multi-factorial health issue caused by viral/bacterial pathogens and stressors linked to the transport and mixing of cattle, negatively impacting the cattle feedlot industry. Common practice during processing at feedlots is administration of antibiotic metaphylaxis to reduce the incidence of BRDc. Nitric oxide (NO) is a naturally occurring nano-molecule with a wide range of physiological attributes. This study evaluated the metaphylactic use of intranasal NO releasing spray (NORS) to control BRDc incidence in calves at low-moderate risk of developing BRDc, arriving at a commercial feedlot as compared to conventional antibiotic metaphylaxis. One thousand and eighty crossbred, multiple-sourced, commingled, commercial, weaned beef calves were screened, enrolled, randomized and treated upon arrival. Animals appearing sick were pulled (from their pen) by blinded pen keepers then assessed for BRDc symptoms; blood samples were taken for haptoglobin quantification and the animals were rescued with an antibiotic. After 35 days both groups showed no significant difference in BRDc incidence (5.2% of animals from NORS group and 3.2% from antibiotic group). Average daily weight gain of animals at day 150 for the NORS cohort was 1.17kg compared to 1.18kg for the antibiotic group (p>0.05). There was no significant difference in mortality in the first 35 days (p=0.7552), however, general mortality over 150 days trended higher in the antibiotic cohort. NORS treatment was shown to be safe, causing neither distress nor adverse effects on the animals. This large randomized controlled study in low-moderate BRDc incidence risk calves demonstrates that NORS treatment, as compared to conventional metaphylactic antibiotics, is non-inferior based on BRDc incidence and other metrics like weight and mortality. These data justify further studies in higher BRDc incidence risk populations to evaluate NORS as an alternative strategy to reduce sub-therapeutic metaphylaxis antibiotic use in beef cattle production.

Randomized, non-inferiority trial comparing a nitric oxide releasing solution with a macrolide antibiotic for control of bovine respiratory disease in beef feedlot calves at high-risk of developing respiratory tract disease

Nitric oxide, a molecule produced in most mammalian cells, has bactericidal and virucidal properties. Nasal instillation of a nitric oxide releasing solution (NORS) on arrival at the feedlot was recently reported as non-inferior to a parenteral injection of a macrolide antibiotic, tilmicosin, for control of bovine respiratory disease (BRD) in cattle at low-to-moderate risk of developing BRD. The objective of this study was to evaluate whether NORS was non-inferior to tilmicosin for control of BRD in cattle at high-risk of developing BRD (the target population for many BRD control programs). High-risk Angus-cross heifers (n=840) were randomly allocated to 2 treatment groups on arrival at a feedlot and received either NORS or tilmicosin for BRD control. Non-inferiority was assessed by calculating the difference in prevalence of heifers diagnosed with BRD during the first 40 d after arrival between NORS and tilmicosin treatment groups. The non-inferiority margin (δ) was set at 8.5%. Thirty-six and 19% of heifers were diagnosed with BRD in the NORS and tilmicosin groups, respectively. Because the lower bound of the 2-sided 95% confidence interval (CI) of the difference in BRD prevalence between the 2 treatment groups (17%; 95% CI=11-23%) was higher than δ, an inferiority of NORS was concluded. Although on-arrival nasal administration of NORS can be viewed as a more rational control strategy than parental injection of antibiotics, further research is needed to improve NORS efficacy before it can be recommended to prevent BRD in high-risk cattle.

Nitric oxide modulates the immunological response of bovine PBMCs in an in vitro BRDc infection model

Bovine respiratory disease complex (BRDc) is a multi-factorial disease, involving both viral and bacterial pathogens, that negatively impacts the cattle feedlot industry. A nitric oxide releasing solution (NORS) has been developed and shown to have potential in the prevention of BRDc. This study investigated the underlying immunological mechanisms through which the nitroslyating agent NORS provides protection against the development of BRDc in susceptible cattle. An in vitro BRDc experimental model was designed using bovine peripheral blood mononuclear cells (PBMCs) which were infected with bovine herpesvirus 1 (BHV-1) and subsequently cultured with lipopolysaccharides (LPS) extracted from Mannheimia haemolytica bacteria. The cells were treated with NORS following viral infection to reflect the timing of administering the NORS treatment in feedlots during initial processing. An expression and protein analysis of key genes involved in the innate immune response was carried out. The BRDc model produced significant increases in gene expression (p<0.01) and protein release (p<0.05) of the proinflammatory cytokines IL-1β and TNF. Treatment with NORS reduced the protein levels of IL-1β (0.39-fold↓) (p<0.05) and TNF (0.48-fold↓) (p<0.01) in the BRDc experimental group when compared against the non-treatment BRDc controls. TLR4 expression, having been significantly reduced under the BRDc experimental conditions (0.33-fold↓) (p<0.05), increased significantly (0.76-fold↑) (p<0.05) following NORS treatment. This study provides evidence suggesting that NO may protect against the development of BRDc by limiting deleterious inflammation while simultaneously increasing TLR4 expression and enhancing the ability of the host to detect and respond to bacterial pathogens.