Mechanism of Ocular Penetration of Lipophilic Drugs from Lipophilic Vehicles.

Since eyedrops have conventionally been formulated in aqueous vehicles, ocular pharmacokinetic studies are generally performed using aqueous buffers to identify physicochemical properties of the drug and the vehicles that influence drug absorption. In recent years, biocompatible lipophilic vehicles are increasingly finding application in ocular drug delivery; however, the mechanism of drug penetration from these non-aqueous vehicles is poorly understood. This study aims to compare ocular penetration of the model lipophilic drug curcumin when incorporated into lipophilic vehicles. To elucidate whether intrinsic solubility in the lipophilic vehicle influences ocular penetration, a curcumin solution and suspension were prepared in medium chain triglycerides (MCT) and squalane, respectively. Ocular penetration and distribution of curcumin from both vehicles was compared and evaluated qualitatively and quantitatively ex vivo. Significantly greater and faster penetration was observed from the squalane suspension than from the MCT solution in all ocular tissues. Our results suggest that the ability of lipophilic drugs to partition out of lipophilic vehicles and into cell membranes, rather than their intrinsic solubility in the lipophilic vehicle, determines the rate and extent of their ocular penetration.

Class-Driven Synergy and Antagonism between a Pseudomonas Phage and Antibiotics.

The ubiquitous bacterial pathogen Pseudomonas aeruginosa is responsible for severe infections in patients with burns, cystic fibrosis, and neutropenia. Biofilm formation gives physical refuge and a protected microenvironment for sessile cells, rendering cure by antibiotics a challenge. Bacteriophages have evolved to prey on these biofilms over millions of years, using hydrolases and depolymerases to penetrate biofilms and reach cellular targets. Here, we assessed how a newly discovered KMV-like phage (ΦJB10) interacts with antibiotics to treat P. aeruginosa more effectively in both planktonic and biofilm forms. By testing representatives of four classes of antibiotics (cephalosporins, aminoglycosides, fluoroquinolones, and carbapenems), we demonstrated class-dependent interactions between ΦJB10 and antibiotics in both biofilm clearance and P. aeruginosa killing. Despite identifying antagonism between some antibiotic classes and ΦJB10 at early time points, all classes showed neutral to favorable interactions with the phage at later time points. In one notable example where the antibiotic alone had poor activity against both biofilm and high-density planktonic cells, we found that addition of ΦJB10 demonstrated synergy and resulted in effective treatment of both. Further, ΦJB10 seemed to act as an adjuvant to several antibiotics, reducing the concentration of antibiotics required to ablate the biofilm. This report shows that phages such as ΦJB10 may be valuable additions to the armamentarium against difficult-to-treat biofilm-based infections.

Effect of various types of extracellular DNA on V. hyugaensis biofilm formation.

Marine bacteria face a constant influx of new extracellular DNA (exDNA) due to the massive viral lysis that occurs in the ocean on a daily basis. Generally, biofilms have shown to be induced by self-secreted exDNA. However, the effect of various types of exDNA with varying lengths, self vs non-self, as well as guanine-cytosine content (GC) content on biofilm formation has not been explored, despite being a critical component of the extracellular polymeric substance. To test the effect of such exDNA on biofilms, a marine bioluminescent bacterium (Vibrio hyugaensis) was isolated from the Sippewissett Salt Marsh, USA, and treated with various types of exDNA. We observed rapid pellicle formation with distinct morphologies only in cultures treated with herring sperm gDNA, another Vibrio spp. gDNA, and an oligomer of 61-80% GC content. With pH measurements before and after the treatment, we observed a positive correlation between biofilm formation and the change to a more neutral pH. Our study highlights the importance of studying DNA-biofilm interaction by carefully examining the physical properties of the DNA and by varying its content, length, and source. Our observation may serve as the basis for future studies that seek to interrogate the molecular explanation for the various types of exDNA and their effects on biofilm formation. IMPORTANCE Bacteria mostly exist as biofilm, a protective niche that promotes protection from the environment and nutrient uptake. By forming these structures, bacteria have caused recalcitrant antibiotic-resistant infections, contamination of dairy and seafood, and fouling equipment in the industry. A critical component that makes up the extracellular polymeric substances, the structural component of a biofilm, is the extracellular DNA secreted by the bacteria found in the biofilm. However, previous studies on DNA and biofilm formation have neglected the unique properties of nucleic acid and its high diversity. Our study aims at disentangling these DNA properties by monitoring their effect at inducing biofilm formation. By varying length, self vs non-self, and GC percentage, we used various microscopy techniques to visualize the structural composition of a Vibrio hyugaensis biofilm. We observed DNA-dependent biofilm stimulation in this organism, a novel function of DNA in biofilm biology.

Metabolic profiling reveals nutrient preferences during carbon utilization in Bacillus species.

The genus Bacillus includes species with diverse natural histories, including free-living nonpathogenic heterotrophs such as B. subtilis and host-dependent pathogens such as B. anthracis (the etiological agent of the disease anthrax) and B. cereus, a cause of food poisoning. Although highly similar genotypically, the ecological niches of these three species are mutually exclusive, which raises the untested hypothesis that their metabolism has speciated along a nutritional tract. Here, we developed a pipeline for quantitative total assessment of the use of diverse sources of carbon for general metabolism to better appreciate the "culinary preferences" of three distinct Bacillus species, as well as related Staphylococcus aureus. We show that each species has widely varying metabolic ability to utilize diverse sources of carbon that correlated to their ecological niches. This approach was applied to the growth and survival of B. anthracis in a blood-like environment and find metabolism shifts from sugar to amino acids as the preferred source of energy. Finally, various nutrients in broth and host-like environments are identified that may promote or interfere with bacterial metabolism during infection.

Targeting of Mammalian Glycans Enhances Phage Predation in the Gastrointestinal Tract.

The human gastrointestinal mucosal surface consists of a eukaryotic epithelium, a prokaryotic microbiota, and a carbohydrate-rich interface that separates them. In the gastrointestinal tract, the interaction of bacteriophages (phages) and their prokaryotic hosts influences the health of the mammalian host, especially colonization with invasive pathobionts. Antibiotics may be used, but they also kill protective commensals. Here, we report a novel phage whose lytic cycle is enhanced in intestinal environments. The tail fiber gene, whose protein product binds human heparan sulfated proteoglycans and localizes the phage to the epithelial cell surface, positions it near its bacterial host, a type of locational targeting mechanism. This finding offers the prospect of developing mucosal targeting phage to selectively remove invasive pathobiont species from mucosal surfaces.IMPORTANCE Invasive pathobionts or microbes capable of causing disease can reside deep within the mucosal epithelium of our gastrointestinal tract. Targeted effective antibacterial therapies are needed to combat these disease-causing organisms, many of which may be multidrug resistant. Here, we isolated a lytic bacteriophage (phage) that can localize to the epithelial surface by binding heparan sulfated glycans, positioning it near its host, Escherichia coli This targeted therapy can be used to selectively remove invasive pathobionts from the gastrointestinal tract, preventing the development of disease.

Phage-Antibiotic Synergy Is Driven by a Unique Combination of Antibacterial Mechanism of Action and Stoichiometry.

The continued rise in antibiotic resistance is precipitating a medical crisis. Bacteriophage (phage) has been hailed as one possible therapeutic option to augment the efficacy of antibiotics. However, only a few studies have addressed the synergistic relationship between phage and antibiotics. Here, we report a comprehensive analysis of phage-antibiotic interaction that evaluates synergism, additivism, and antagonism for all classes of antibiotics across clinically achievable stoichiometries. We combined an optically based real-time microtiter plate readout with a matrix-like heat map of treatment potencies to measure phage and antibiotic synergy (PAS), a process we term synography. Phage-antibiotic synography was performed against a pandemic drug-resistant clonal group of extraintestinal pathogenic Escherichia coli (ExPEC) with antibiotic levels blanketing the MIC across seven orders of viral titers. Our results suggest that, under certain conditions, phages provide an adjuvating effect by lowering the MIC for drug-resistant strains. Furthermore, synergistic and antagonistic interactions are highly dependent on the mechanism of bacterial inhibition by the class of antibiotic paired to the phage, and when synergism is observed, it suppresses the emergence of resistant cells. Host conditions that simulate the infection environment, including serum and urine, suppress PAS in a bacterial growth-dependent manner. Lastly, two different related phages that differed in their burst sizes produced drastically different synograms. Collectively, these data suggest lytic phages can resuscitate an ineffective antibiotic for previously resistant bacteria while also synergizing with antibiotics in a class-dependent manner, processes that may be dampened by lower bacterial growth rates found in host environments.IMPORTANCE Bacteriophage (phage) therapy is a promising approach to combat the rise of multidrug-resistant bacteria. Currently, the preferred clinical modality is to pair phage with an antibiotic, a practice thought to improve efficacy. However, antagonism between phage and antibiotics has been reported, the choice of phage and antibiotic is not often empirically determined, and the effect of the host factors on the effectiveness is unknown. Here, we interrogate phage-antibiotic interactions across antibiotics with different mechanisms of action. Our results suggest that phage can lower the working MIC for bacterial strains already resistant to the antibiotic, is dependent on the antibiotic class and stoichiometry of the pairing, and is dramatically influenced by the host microenvironment.

Tailored Antibacterials and Innovative Laboratories for Phage (Φ) Research: Personalized Infectious Disease Medicine for the Most Vulnerable At-Risk Patients.

Mutation is the most powerful driver of change for life on Earth. Pathogenic bacteria utilize mutation as a means to survive strong live-die selective pressures generated by chemical antibiotics. As such, the traditional drug-making pipeline, characterized by significant financial and time investment, is insufficient to keep pace with the rapid evolution of bacterial resistance to structurally fixed and chemically unmalleable antibacterial compounds. In contrast, the genetic diversity and adaptive mutability of the bacteriophage can be leveraged to not only overcome resistance but also used for the development of enhanced traits that increase lytic potential and therapeutic efficacy in relevant host microenvironments. This is the fundamental premise behind Baylor College of Medicine's Tailored Antibacterials and Innovative Laboratories for Phage (Φ) Research (TAILΦR) initiative. In this perspective, we outline the concept, structure, and process behind TAILΦR's attempt to generate a personalized therapeutic phage that addresses the most clinically challenging of bacterial infections.

Constructing and Characterizing Bacteriophage Libraries for Phage Therapy of Human Infections.

Phage therapy requires libraries of well-characterized phages. Here we describe the generation of phage libraries for three target species: Escherichia coli, Pseudomonas aeruginosa, and Enterobacter cloacae. The basic phage characteristics on the isolation host, sequence analysis, growth properties, and host range and virulence on a number of contemporary clinical isolates are presented. This information is required before phages can be added to a phage library for potential human use or sharing between laboratories for use in compassionate use protocols in humans under eIND (emergency investigational new drug). Clinical scenarios in which these phages can potentially be used are discussed. The phages presented here are currently being characterized in animal models and are available for eINDs.

A Longitudinal Study on the Prevalence and Risk Factors for Depression and Anxiety, Quality of Life, and Clinical Outcomes in Incident Peritoneal Dialysis Patients.

BACKGROUND: Starting dialysis is an important life event. The prevalence and evolution of psychological symptoms at commencement of long-term dialysis is unclear. We examined the prevalence of and risk factors for depression and anxiety, and the quality of life (QOL) of incident peritoneal dialysis (PD) patients, and also the change of these parameters in the first year of PD in relation to clinical outcomes under the PD-first policy. METHODS: All patients commencing long-term PD from March 2011 to April 2015 were asked to complete the Hospital Anxiety and Depression Scale (HADS), World Health Organization Quality of Life-BREF and the Kidney Disease Quality of Life Instrument Short Form questionnaire. Patient demographics and the incidence of hospitalization, peritonitis, exit-site infection, and all-cause mortality were studied. The HADS was repeated after 9 - 12 months. RESULTS: A high depression score was present in 39.6% of 191 patients at commencement of PD and was more common in diabetes patients (odds ratio [OR] 2.03, 95% confidence interval [CI] 1.09 - 3.81). A high anxiety score was present in 23.6%, and the risk factors included younger age (OR 0.96 per year, 95% CI 0.94 - 0.99) and diabetes (OR 2.59, 95% CI 1.20 - 5.57). Both high depression and anxiety scores were associated with an inferior QOL, overall and across most QOL domains. Depression and anxiety symptoms did not change in the first year of PD and were not associated with short-term clinical outcomes. CONCLUSIONS: High depression and anxiety scores were prevalent in incident PD patients where PD-first policy is adopted and were associated with inferior QOL. There was no improvement after 1 year of PD. The impact of strategic interventions targeting patient groups at risk such as those with diabetes or of younger age warrants further investigation.