Comparing Methods to Genetically Engineer Bacteriophage and Increase Host Range.

INTRODUCTION: Antibacterial resistance is an emerging problem in military medicine. Disruptions to the health care systems in war-torn countries that result from ongoing conflict can potentially exacerbate this problem and increase the risk to U.S. forces in the deployed environment. Therefore, novel therapies are needed to mitigate the impact of these potentially devastating infections on military operations. Bacteriophages are viruses that infect and kill bacteria. They can be delivered as therapeutic agents and offer a promising alternative to traditional antibiotic chemotherapy. There are several potential benefits to their use, including high specificity and comparative ease of use in the field setting. However, the process of engineering phages for military medical applications can be a laborious and time-consuming endeavor. This review examines available techniques and compares their efficacy. MATERIALS AND METHODS: This review evaluates the scientific literature on the development and application of four methods of bacteriophage genome engineering and their consideration in the context of military applications. Preffered Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed for a systematic review of available literature that met criteria for analysis and inclusion. The research completed for this review article originated from the United States Military Academy's library "Scout" search engine, which compiles results from 254 available databases (including PubMed, Google Scholar, and SciFinder). Particular attention was focused on identifying useful mechanistic insight into the nature of the engineering technique, the ease of use, and the applicability of the technique to countering the problem of antimicrobial resistance in the military setting. RESULTS: A total of 52 studies were identified that met inclusion criteria following PRISMA guidelines. The bioengineering techniques analyzed included homologous recombination (12 articles), in vivo recombineering (9 articles), bacteriophage recombineering of electroporated DNA (7 articles), and the CRISPR-Cas system (10 articles). Rates of success and fidelity varied across each platform, and comparative benefits and drawbacks are considered. CONCLUSIONS: Each of the phage engineering techniques addressed herein varies in amount of effort and overall success rate. CRISPR-Cas-facilitated modification of phage genomes presents a highly efficient method that does not require a lengthy purification and screening process. It therefore appears to be the method best suited for military medical applications.

Disruption of Biofilm by Bacteriophages in Clinically Relevant Settings.

INTRODUCTION: Antibiotic-resistant bacteria are a growing threat to civilian and military health today. Although infections were once easily treatable by antibiotics and wound cleaning, the frequent mutation of bacteria has created strains impermeable to antibiotics and physical attack. Bacteria further their pathogenicity because of their ability to form biofilms on wounds, medical devices, and implant surfaces. Methods for treating biofilms in clinical settings are limited, and when formed by antibiotic-resistant bacteria, can generate chronic infections that are recalcitrant to available therapies. Bacteriophages are natural viral predators of bacteria, and their ability to rapidly destroy their host has led to increased attention in potential phage therapy applications. MATERIALS AND METHODS: The present article sought to address a knowledge gap in the available literature pertaining to the usage of bacteriophage in clinically relevant settings and the resolution of infections particular to military concerns. PRISMA guidelines were followed for a systematic review of available literature that met the criteria for analysis and inclusion. The research completed for this review article originated from the U.S. Military Academy's library "Scout" search engine, which complies results from 254 available databases (including PubMed, Google Scholar, and SciFinder). The search criteria included original studies that employed bacteriophage use against biofilms, as well as successful phage therapy strategies for combating chronic bacterial infections. We specifically explored the use of bacteriophage against antibiotic- and treatment-resistant bacteria. RESULTS: A total of 80 studies were identified that met the inclusion criteria following PRISMA guidelines. The application of bacteriophage has been demonstrated to robustly disrupt biofilm growth in wounds and on implant surfaces. When traditional therapies have failed to disrupt biofilms and chronic infections, a combination of these treatments with phage has proven to be effective, often leading to complete wound healing without reinfection. CONCLUSIONS: This review article examines the available literature where bacteriophages have been utilized to treat biofilms in clinically relevant settings. Specific attention is paid to biofilms on implant medical devices, biofilms formed on wounds, and clinical outcomes, where phage treatment has been efficacious. In addition to the clinical benefit of phage therapies, the military relevance and treatment of combat-related infections is also examined. Phages offer the ability to expand available treatment options in austere environments with relatively low cost and effort, allowing the impacted warfighter to return to duty quicker and healthier.

Disruption of l-Rhamnose Biosynthesis Results in Severe Growth Defects in Streptococcus mutans.

The rhamnose-glucose cell wall polysaccharide (RGP) of Streptococcus mutans plays a significant role in cell division, virulence, and stress protection. Prior studies examined function of the RGP using strains carrying deletions in the machinery involved in RGP assembly. In this study, we explored loss of the substrate for RGP, l-rhamnose, via deletion of rmlD (encoding the protein responsible for the terminal step in l-rhamnose biosynthesis). We demonstrate that loss of rhamnose biosynthesis causes a phenotype similar to strains with disrupted RGP assembly (ΔrgpG and ΔrgpF strains). Deletion of rmlD not only caused a severe growth defect under nonstress growth conditions but also elevated susceptibility of the strain to acid and oxidative stress, common conditions found in the oral cavity. A genetic complement of the ΔrmlD strain completely restored wild-type levels of growth, whereas addition of exogenous rhamnose did not. The loss of rhamnose production also significantly disrupted biofilm formation, an important aspect of S. mutans growth in the oral cavity. Further, we demonstrate that loss of either rmlD or rgpG results in ablation of rhamnose content in the S. mutans cell wall. Taken together, these results highlight the importance of rhamnose production in both the fitness and the ability of S. mutans to overcome environmental stresses.IMPORTANCEStreptococcus mutans is a pathogenic bacterium that is the primary etiologic agent of dental caries, a disease that affects billions yearly. Rhamnose biosynthesis is conserved not only in streptococcal species but in other Gram-positive, as well as Gram-negative, organisms. This study highlights the importance of rhamnose biosynthesis in RGP production for protection of the organism against acid and oxidative stresses, the two major stressors that the organism encounters in the oral cavity. Loss of RGP also severely impacts biofilm formation, the first step in the onset of dental caries. The high conservation of the rhamnose synthesis enzymes, as well as their importance in S. mutans and other organisms, makes them favorable antibiotic targets for the treatment of disease.

Streptococcus mutans requires mature rhamnose-glucose polysaccharides for proper pathophysiology, morphogenesis and cellular division.

The cell wall of Gram-positive bacteria has been shown to mediate environmental stress tolerance, antibiotic susceptibility, host immune evasion and overall virulence. The majority of these traits have been demonstrated for the well-studied system of wall teichoic acid (WTA) synthesis, a common cell wall polysaccharide among Gram-positive organisms. Streptococcus mutans, a Gram-positive odontopathogen that contributes to the enamel-destructive disease dental caries, lacks the capabilities to generate WTA. Instead, the cell wall of S. mutans is highly decorated with rhamnose-glucose polysaccharides (RGP), for which functional roles are poorly defined. Here, we demonstrate that the RGP has a distinct role in protecting S. mutans from a variety of stress conditions pertinent to pathogenic capability. Mutant strains with disrupted RGP synthesis failed to properly localize cell division complexes, suffered from aberrant septum formation and exhibited enhanced cellular autolysis. Surprisingly, mutant strains of S. mutans with impairment in RGP side chain modification grew into elongated chains and also failed to properly localize the presumed cell wall hydrolase, GbpB. Our results indicate that fully mature RGP has distinct protective and morphogenic roles for S. mutans, and these structures are functionally homologous to the WTA of other Gram-positive bacteria.

Acanthamoeba encystment: multifactorial effects of buffers, biocides, and demulcents present in contact lens care solutions.

PURPOSE: To determine whether agents which are purportedly capable of inducing encystment of Acanthamoeba can recapitulate the signal when tested in differing formulations. METHODS: In accordance with the International Standard ISO 19045, Acanthamoeba castellanii ATCC 50370 trophozoites were cultured in antibiotic-free axenic medium, treated with test solutions, and encystment rates plus viability were measured via bright field and fluorescent microscopy. Test solutions included phosphate-buffered saline (PBS), borate-buffered saline, biguanide- and hydrogen peroxide (H2O2)-based biocides, propylene glycol (PG) and povidone (POV) ophthalmic demulcents, and one-step H2O2-based contact lens disinfection systems. RESULTS: Only PBS solutions with 0.25 ppm polyaminopropyl biguanide (PAPB) and increasing concentrations of PG and POV stimulated A. castellanii encystment in a dose-dependent manner, whereas PBS solutions containing 3% H2O2 and increasing concentrations of PG and POV did not stimulate encystment. Borate-buffered saline and PBS/citrate solutions containing PG also did not stimulate encystment. In addition, no encystment was observed after 24 hours, 7 days, or 14 days of exposures of trophozoites to one-step H2O2 contact lens disinfection products or related solutions. CONCLUSION: The lack of any encystment observed when trophozoites were treated with existing or new one-step H2O2 contact lens care products, as well as when trophozoites were exposed to various related test solutions, confirms that Acanthamoeba encystment is a complex process which depends upon simultaneous contributions of multiple factors including buffers, biocides, and demulcents.

Glucose 6-phosphate dehydrogenase is regulated through c-Src-mediated tyrosine phosphorylation in endothelial cells.

OBJECTIVE: Glucose 6-phosphate dehydrogenase (G6PD) maintains cellular NADPH levels, which are essential for cellular functions, such as vascular endothelial growth factor (VEGF)-induced angiogenesis. The molecular mechanisms regulating G6PD in angiogenesis are not fully understood. Because tyrosine phosphorylation is a key regulatory pathway for VEGF-mediated endothelial cell (EC) responses, we investigated tyrosine phosphorylation of G6PD and the role of the nonreceptor tyrosine kinase Src. METHODS AND RESULTS: VEGF increased G6PD membrane translocation as measured by a plasma membrane sheet assay, whereas tyrosine kinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-d] pyramidine) decreased G6PD translocation by 100%. Furthermore, G6PD tyrosine phosphorylation and plasma membrane activity were increased by VEGF. In resting ECs, tyrosine kinase inhibitors PP2 and herbimycin A decreased basal G6PD activity by approximately 25%, whereas transfection with kinase inactive Src (KD-Src) decreased basal activity by approximately 30%. In mouse embryonic fibroblasts, Src-deficient (SYF) cells showed approximately 22% lower basal G6PD activity than Src-expressing S(+)YF cells. In addition, Src directly phosphorylated G6PD assayed by in vitro kinase assay. In ECs transfected with the G6PD mutants Y428F, Y507F (presumptive sites for Src-phosphorylation) or double mutant Y428F/Y507F, G6PD tyrosine phosphorylation was significantly decreased. Finally, G6PD tyrosine mutants (Y428F, Y507F, and Y428F/Y507F) decreased VEGF-mediated Akt phosphorylation and EC migration. CONCLUSIONS: G6PD activity and membrane association are regulated by Src-mediated tyrosine phosphorylation, which contributes to VEGF-mediated cellular responses in EC.