Combinatorial materials research applied to the development of new surface coatings IV. A high-throughput bacterial biofilm retention and retraction assay for screening fouling-release performance of coatings

Abstract

A high-throughput bacterial biofilm retention screening method has been augmented to facilitate the rapid analysis and down-selection of fouling-release coatings for identification of promising candidates. Coatings were cast in modified 24-well tissue culture plates and inoculated with the marine bacterium Cytophaga lytica for attachment and biofilm growth. Biofilms retained after rinsing with deionised water were dried at ambient laboratory conditions. During the drying process, retained biofilms retracted through a surface de-wetting phenomenon on the hydrophobic silicone surfaces. The retracted biofilms were stained with crystal violet, imaged, and analysed for percentage coverage. Two sets of experimental fouling-release coatings were analysed with the high-throughput biofilm retention and retraction assay (HTBRRA). The first set consisted of a series of model polysiloxane coatings that were systematically varied with respect to ratios of low and high MW silanol-terminated PDMS, level of cross-linker, and amount of silicone oil. The second set consisted of cross-linked PDMS-polyurethane coatings varied with respect to the MW of the PDMS and end group functionality. For the model polysiloxane coatings, HTBRRA results were compared to data obtained from field immersion testing at the Indian River Lagoon at the Florida Institute of Technology. The percentage coverage calculations of retracted biofilms correlated well to barnacle adhesion strength in the field (R² = 0.82) and accurately identified the best and poorest performing coating compositions. For the cross-linked PDMS-polyurethane coatings, the HTBRRA results were compared to combinatorial pseudobarnacle pull-off adhesion data and good agreement in performance was observed. Details of the developed assay and its implications in the rapid discovery of new fouling-release coatings are discussed.     

In vivo effects of anacetrapib on preβ HDL: improvement in HDL remodeling without effects on cholesterol absorption

Cholesteryl ester transfer protein (CETP) transfers cholesteryl ester and triglyceride between HDL and apoB-containing lipoproteins. Anacetrapib (ANA), a reversible inhibitor of CETP, raises HDL cholesterol and lowers LDL cholesterol in dyslipidemic patients. We previously demonstrated that ANA increases macrophage-to-feces reverse cholesterol transport and fecal cholesterol excretion in hamsters, and increased preβ HDL-dependent cholesterol efflux via ABCA1 in vitro. However, the effects of ANA on in vivo preβ HDL have not been characterized. In vitro, ANA inhibited the formation of preβ, however in ANA-treated dyslipidemic hamsters, preβ HDL levels (measured by two-dimensional gel electrophoresis) were increased, in contrast to in vitro findings. Because changes in plasma preβ HDL have been proposed to potentially affect markers of cholesterol absorption with other CETP inhibitors, a dual stable isotope method was used to directly measure cholesterol absorption in hamsters. ANA treatment of hamsters (on either dyslipidemic or normal diet) had no effect on cholesterol absorption, while dalcetrapib-treated hamsters displayed an increase in cholesterol absorption. Taken together, these data support the notion that ANA promotes preβ HDL functionality in vivo, with no effects on cholesterol absorption.     

Long-term calorie restriction has minimal impact on brain metabolite and fatty acid profiles in aged rats on a Western-style diet

The effect of long-term calorie restriction (CR) on metabolites, fatty acid profiles and energy substrate transporter expression in the brain was assessed in aged rats. Three groups of male Sprague–Dawley rats were studied: (i) a 2 month old ad libitum-fed (2AL group), (ii) a 19 month old ad libitum-fed (19AL group), and (iii) a 19 month old group subjected to 40% CR from the age of 7.5 to 19 months (19CR group). The diet contained high sucrose and low n-3 polyunsaturated fatty acids (PUFA) so as to imitate a Western-style diet. High resolution magic angle spinning-¹H NMR showed an effect of aging on brain cortex metabolites compared to 2AL rats, the largest differences being for myo-inositol (+251% and +181%), lactate (+203% and +188%), β-hydroxybutyrate (+176% and +618%) and choline (+148% and +120%), in 19AL and 19 CR rats, respectively. However, brain metabolites did not differ between the 19AL and 19CR groups. Cortex fatty acid profiles showed that n-3 PUFA were 35–47% lower but monounsaturated fatty acids were 40–52% higher in 19AL and 19CR rats compared to 2AL rats. Brain microvessel glucose transporter (GLUT1) was 68% higher in 19AL rats than in 2AL rats, while the monocarboxylate transporter, MCT1, was 61% lower in 19CR rats compared to 19AL rats. We conclude that on a high-sucrose, low n-3 PUFA diet, the brain of aged AL rats had higher metabolites and microvessel GLUT1 expression compared to 2AL rats. However, long-term CR in aged rats did not markedly change brain metabolite or fatty acid profile, but did reduce brain microvessel MCT1 expression.     

Development of an in vivo active,dual EP1 and EP3 selective antagonist based on a novel acyl sulfonamide bioisostere

Recent preclinical studies demonstrate a role for the prostaglandin E₂ (PGE₂) subtype 1 (EP1) receptor in mediating, at least in part, the pathophysiology of hypertension and diabetes mellitus. A series of amide and N-acylsulfonamide analogs of a previously described picolinic acid-based human EP1 receptor antagonist (7) were prepared. Each analog had improved selectivity at the mouse EP1 receptor over the mouse thromboxane receptor (TP). A subset of analogs gained affinity for the mouse PGE₂ subtype 3 (EP3) receptor, another potential therapeutic target. One analog (17) possessed equal selectivity for EP1 and EP3, displayed a sufficient in vivo residence time in mice, and lacked the potential for acyl glucuronide formation common to compound 7. Treatment of mice with 17 significantly attenuated the vasopressor activity resulting from an acute infusion of EP1 and EP3 receptor agonists. Compound 17 represents a potentially novel therapeutic in the treatment of hypertension and diabetes mellitus.     

Local Inflammation Exacerbates the Severity of Staphylococcus aureus Skin Infection

Staphylococcus aureus is the leading cause of skin infections. In a mouse model of S. aureus skin infection, we found that lesion size did not correlate with bacterial burden. Athymic nude mice had smaller skin lesions that contained lower levels of myeloperoxidase, IL-17A, and CXCL1, compared with wild type mice, although there was no difference in bacterial burden. T cell deficiency did not explain the difference in lesion size, because TCR βδ (-/-) mice did not have smaller lesions, and adoptive transfer of congenic T cells into athymic nude mice prior to infection did not alter lesion size. The differences observed were specific to the skin, because mortality in a pneumonia model was not different between wild type and athymic nude mice. Thus, the clinical severity of S. aureus skin infection is driven by the inflammatory response to the bacteria, rather than bacterial burden, in a T cell independent manner.     

Respiratory microbiota resistance and resilience to pulmonary exacerbation and subsequent antimicrobial intervention

Pulmonary symptoms in cystic fibrosis (CF) begin in early life with chronic lung infections and concomitant airway inflammation leading to progressive loss of lung function. Gradual pulmonary function decline is interspersed with periods of acute worsening of respiratory symptoms known as CF pulmonary exacerbations (CFPEs). Cumulatively, CFPEs are associated with more rapid disease progression. In this study multiple sputum samples were collected from adult CF patients over the course of CFPEs to better understand how changes in microbiota are associated with CFPE onset and management. Data were divided into five clinical periods: pre-CFPE baseline, CFPE, antibiotic treatment, recovery, and post-CFPE baseline. Samples were treated with propidium monoazide prior to DNA extraction, to remove the impact of bacterial cell death artefacts following antibiotic treatment, and then characterised by 16S rRNA gene-targeted high-throughput sequencing. Partitioning CF microbiota into core and rare groups revealed compositional resistance to CFPE and resilience to antibiotics interventions. Mixed effects modelling of core microbiota members revealed no significant negative impact on the relative abundance of Pseudomonas aeruginosa across the exacerbation cycle. Our findings have implications for current CFPE management strategies, supporting reassessment of existing antimicrobial treatment regimens, as antimicrobial resistance by pathogens and other members of the microbiota may be significant contributing factors.     

β-arrestin 2 attenuates cardiac dysfunction in polymicrobial sepsis through gp130 and p38

Sepsis is an exaggerated systemic inflammatory response to persistent bacteria infection with high morbidity and mortality rate clinically. β-arrestin 2 modulates cell survival and cell death in different systems. However, the effect of β-arrestin 2 on sepsis-induced cardiac dysfunction is not yet known. Here, we show that β-arrestin 2 overexpression significantly enhances animal survival following cecal ligation and puncture (CLP)-induced sepsis. Importantly, overexpression of β-arrestin 2 in mice prevents CLP-induced cardiac dysfunction. Also, β-arrestin 2 overexpression dramatically attenuates CLP-induced myocardial gp130 and p38 mitogen-activated protein kinase (MAPK) phosphorylation levels following CLP. Therefore, β-arrestin 2 prevents CLP-induced cardiac dysfunction through gp130 and p38. These results suggest that modulation of β-arrestin 2 might provide a novel therapeutic approach to prevent cardiac dysfunction in patients with sepsis.     

Comparison of laboratory and field testing performance evaluations of siloxane-polyurethane fouling-release marine coatings

A series of eight novel siloxane-polyurethane fouling-release (FR) coatings were assessed for their FR performance in both the laboratory and in the field. Laboratory analysis included adhesion assessments of bacteria, microalgae, macroalgal spores, adult barnacles and pseudobarnacles using high-throughput screening techniques, while field evaluations were conducted in accordance with standardized testing methods at three different ocean testing sites over the course of six-months exposure. The data collected were subjected to statistical analysis in order to identify potential correlations. In general, there was good agreement between the laboratory screening assays and the field assessments, with both regimes clearly distinguishing the siloxane-polyurethane compositions comprising monofunctional poly(dimethyl siloxane) (PDMS) (m-PDMS) as possessing superior, broad-spectrum FR properties compared to those prepared with difunctional PDMS (d-PDMS). Of the seven laboratory screening techniques, the Cellulophaga lytica biofilm retraction and reattached barnacle (Amphibalanus amphitrite) adhesion assays were shown to be the most predictive of broad-spectrum field performance.