Current Pharmaceutical Biotechnology - Volume 11, Issue 1, 2010

Bacteriophages (phages) are the viruses of bacteria and phage therapy is the application of phages to control or eliminate bacteria and their infections. Phage therapy holds great promise as a means of augmenting the use of chemical antibiotics to control bacteria, including antibiotic-resistant bacteria or under circumstances where antibiotic treatment is less safe or otherwise undesirable. Applications include treatment of bacterial infections of humans, animals, plants, and even fungi (e.g., mushrooms); removal of pathogenic or spoilage bacteria from foods; general disinfection; and disruption of biofilms. Biofilms otherwise can interfere with industrial processes and/or serve as reservoirs of infection, such as within catheters or dialysis machines. The major strengths of phages as antibacterials is their ease of isolation and relative safety while their major disadvantage (though also an advantage) is their narrow spectrum of activity, i.e. host range. The development of an effective phage therapy protocol is a multi-step process. Difficulties at any of these steps can hinder what otherwise can be strengths of using phages as therapeutic agents. These development steps include phage isolation against a specific bacterial strain; testing for activity against that strain as well as against related bacteria; screening for undesirable traits such as the potential to enhance bacterial virulence; growth to high densities; purification away from especially toxins present as residues within bacterial lysates; stabilization for storage; and delivery of viable phages to target bacteria. Ideally, once delivered, phages will survive, not overly stimulate animal immune systems, both adsorb and kill target bacteria, and, in most cases, increase in density and/or spread spatially to reach otherwise minimally accessible bacterial targets. Phages also can be modified using genetic engineering techniques to increase useful characteristics or to decrease potentially detrimental properties. A major concern in the development of phage therapies is regulatory, especially in ways phages differ from standard therapeutic agents. In particular, an ideal environment for phage therapy efficacy appears to be one in which phages may be matched (in terms of host range) to specific bacterial pathogens, evolved toward greater breadth in host range, and/or used as a mixture of multiple phages (a cocktail). Cocktails collectively possess sufficient host-range breadth to treat either a high diversity of pathogens of a specific type or multiple pathogens that can be present as a mixed infection. Phages also are complex chemical entities that are difficult to prevent from evolving. Together these characteristics make phages, at best, simply different from standard chemotherapeutics. At worst, held to the same standards as chemical drugs, phages potentially may be more expensive to move through the regulatory process, which is quite the contrast to a key benefit of phage therapy in places where it already has been approved for human use, such as in the Republic of Georgia: A relatively low cost. Overall, I am guardedly optimistic that phages may come to be increasingly employed as biocontrol agents in a variety of contexts, ranging from agricultural to medical. This special topic issue of Current Pharmaceutical Biotechnology explores the potential of phage therapy within a variety of contexts, taking an especially ‘Nuts and Bolts’ approach i.e., a practical, working guide towards considering what strategies should be most efficacious given real-world application.

Phage therapy is the use of bacteriophages—viruses that use bacteria as their host cells—as biocontrol agents of bacteria. Currently, phage therapy is garnering renewed interest as bacterial resistance to antibiotics becomes widespread. Historically, phage therapy was largely abandoned in the West in the 1940s due to the advent of chemical antibiotics, and the unreliability of phage-based treatments when compared to antibiotics. The choice of phage strain and the methods of phage preparation are now thought to have been critical to the success or failure of phage therapy trials. Insufficiently virulent phages, especially against actual target bacteria, allow bacteria to survive treatment while poorly prepared phage stocks, even if of sufficiently virulent phages, lack the numbers of viable phages required for adequate treatment. In this review we discuss the factors that determine the methods of isolation, analysis, and identification of phage species for phage therapy. We go on to discuss the various methods available for purifying phages as well as considerations of the degree of purification which is sufficient for various applications. Lastly, we review the current practices used to prepare commercial phage therapy products.

Phage therapy is the application of phages to bodies, substances, or environments to effect the biocontrol of pathogenic or nuisance bacteria. To be effective, phages, minimally, must be capable of attaching to bacteria (adsorption), killing those bacteria (usually associated with phage infection), and otherwise surviving (resisting decay) until they achieve attachment and subsequent killing. While a strength of phage therapy is that phages that possess appropriate properties can be chosen from a large diversity of naturally occurring phages, a more rational approach to phage therapy also can include post-isolation manipulation of phages genetically, phenotypically, or in terms of combining different products into a single formulation. Genetic manipulation, especially in these modern times, can involve genetic engineering, though a more traditional approach involves the selection of spontaneously occurring phage mutants during serial transfer protocols. While genetic modification typically is done to give rise to phenotypic changes in phages, phage phenotype alone can also be modified in vitro, prior to phage application for therapeutic purposes, as for the sake of improving phage lethality (such as by linking phage virions to antibacterial chemicals such as chloramphenicol) or survival capabilities (e.g., via virion PEGylation). Finally, phages, both naturally occurring isolates or otherwise modified constructs, can be combined into cocktails which provide collectively enhanced capabilities such as expanded overall host range. Generally these strategies represent different routes towards improving phage therapy formulations and thereby efficacy through informed design.

Phage therapy—application of bacteria-specific viruses to reduce densities of pathogenic or nuisance bacteria— is a two-step process involving phage penetration to target bacteria followed by bacteria killing. Any analysis of these steps is inherently ecological as they represent phage-environment interactions, i.e., between phages and bacteria as well as between phages and body tissues. In considering phages more generically, as selectively toxic antibacterial agents employed to treat bacterial infections, the term “ecology” may be fairly cleanly replaced with the term “pharmacology”. Pharmacology, in turn, may be distinguished into two major components: pharmacokinetics and pharmacodynamics. Pharmacokinetics is explicitly a description of the body's impact on a drug (e.g., movement through and between body compartments) whereas pharmacodynamics is a description of a drug's impact on the body. “Body” includes both body tissues and microbial flora, so an important component of antibacterial pharmacodynamics is inhibition of the growth of target bacteria. Our guiding premise is that phage therapy may be rationally improved through a better understanding of phage pharmacokinetics and pharmacodynamics. Our primary conclusions are (i) that the principle advantages of phages, over antibiotics, are the former's relative safety and ease of discovery; (ii) that phage therapy efficacy is highly dependent on attaining relatively high phage “killing titers”; (iii) that attainment of sufficient titers solely via in situ phage replication should, in some or many circumstances, not be counted upon; and (iv) that phage replication nonetheless may provide a “margin of safety” toward attaining phage therapy efficacy.

Bacteria cause a number of economically important plant diseases. Bacterial outbreaks are generally problematic to control due to lack of effective bactericides and to resistance development. Bacteriophages have recently been evaluated for controlling a number of phytobacteria and are now commercially available for some diseases. Major challenges of agricultural use of phages arise from the inherent diversity of target bacteria, high probability of resistance development, and weak phage persistence in the plant environment. Approaches for resistance management - by applying phage mixtures and host-range mutant phages and, for increasing residual activity, by employing protective formulations, avoiding sunlight, and utilizing propagating bacterial strains - resulted in better efficacy and reliability. Deployment of phage therapy as part of an integrated disease management strategy, which includes the use of genetic control, cultural control, biological control, and chemical control, also has been investigated and will likely increase in the future.

The use of phage or phage products in food production has recently become an option for the food industry as a novel method for biocontrol of unwanted pathogens, enhancing the safety of especially fresh and ready-to-eat food products. While it can be expected that many more phage products currently under development might become available in the future, several questions may be raised concerning the use of such products, regarding both immediate and long-term efficacy, consumer safety, and application methods. The available evidence suggests that, with a few caveats, safety concerns have been satisfactorily addressed. Answers concerning efficacy are more complex, depending on particular applications or the target pathogens. To ensure long-term efficacy beyond what can be tested on a laboratory scale, food safety concepts employing phages will have to be well-thought out and may involve rotation schemes as used with bacterial starter cultures, the use of phage cocktails, or application of phages combined with other antimicrobials. This review will discuss these issues on the basis of the available literature as well as providing an outlook on the potential of phages in future applications.

Phage therapy is the application of bacteria-specific viruses with the goal of reducing or eliminating pathogenic or nuisance bacteria. While phage therapy has become a broadly relevant technology, including veterinary, agricultural, and food microbiology applications, it is for the treatment or prevention of human infections that phage therapy first caught the world's imagination - see, especially, Arrowsmith by Sinclair Lewis (1925) - and which today is the primary motivator of the field. Nonetheless, though the first human phage therapy took place in the 1920s, by the 1940s the field, was in steep decline despite early promise. The causes were at least three-fold: insufficient understanding among researchers of basic phage biology; over exuberance, which led, along with ignorance, to carelessness; and the advent of antibiotics, an easier to handle as well as highly powerful category of antibacterials. The decline in phage therapy was neither uniform nor complete, especially in the former Soviet Republic of Georgia, where phage therapy traditions and practice continue to this day. In this review we strive toward three goals: 1. To provide an overview of the potential of phage therapy as a means of treating or preventing human diseases; 2. To explore the phage therapy state of the art as currently practiced by physicians in various pockets of phage therapy activity around the world, including in terms of potential commercialization; and 3. To avert a recapitulation of phage therapy's early decline by outlining good practices in phage therapy practice, experimentation, and, ultimately, commercialization.

The conversion of prion protein (PrP) from the monomeric cellular isoform to the oligomeric pathological isoform is a crucial event in the pathogenesis of prion diseases. To investigate oligomer formation of PrP, enhanced green fluorescent protein (EGFP)-tagged PrP (EGFP-PrP) without the glycosylphosphatidylinositol (GPI) anchor was prepared and the oligomerization of EGFP-PrP induced by sodium dodecyl sulphate (SDS) was monitored by fluorescence correlation spectroscopy (FCS). The FCS analysis indicated that soluble oligomers were formed at 0.011% SDS. Furthermore, the combination of fluorescence cross-correlation spectroscopy (FCCS) and a panel of anti-PrP monoclonal antibodies (mAbs) revealed the conformational changes in PrP. Our studies provide a method to analyze conformational changes of proteins in solution.

We examined the photophysical properties of the new near infrared (NIR) fluorescent label SeTau-665 on a plasmonic platform of self- assembled colloidal structures (SACS) of silver prepared on a semitransparent silver film. A SeTau-665 immunoassay was performed on this platform and a control glass slide. The fluorescence properties of this label substantially change due to plasmonic interactions. While the average brightness increase of SeTau 665 in ensemble measurements was about 70-fold, fluorescence enhancements up to four-hundred times were observed on certain “hot spots” for single molecule measurements. The intensity increase is strongly correlated with a simultaneous decrease in fluorescence lifetime in these “hot spots”. The large increase in brightness allows the reduction of the excitation power resulting in a reduced background and increased photostability. The remarkable fluorescence enhancements observed for SeTau 665 on our plasmonic platform should allow to substantially improve single molecule detection and to reduce the detection limits in sensing devices.

Mammalian cell cultivation plays a great role in producing protein therapeutics in the last decades. Many engineering parameters are considered for optimization during process development in mammalian cell cultivation, only shear and mixing are especially highlighted in this paper. It is believed that shear stress due to agitation has been over-estimated to damage cells, but shear may result in nonlethal physiological responses. There is no cell damage in the regions where bubbles form, break up and coalescence, but shear stress becomes significant in the wake of rising bubbles and causes great damage to cells in bubble burst regions. Mixing is not sufficient to provide homogeneous dissolved oxygen tension, pH, CO2 and nutrients in large-scale bioreactors, which can bring severe problems for cell growth, product formation and process control. Scale-down reactors have been developed to address mixing and shear problems for parallel operations. Engineering characterization in conventional and recently developed scale-down bioreactors has been briefly introduced. Process challenges for cultivation of industrial cell lines in high cell densities as well as cultivation of stem cells and other human cells for regenerative medicine, tissue engineering and gene therapy are prospected. Important techniques, such as micromanipulation and nanomanipulation (optical tweezers) for single cell analysis, computational fluid dynamics (CFD) for shear and mixing characterization, and miniaturized bioreactors, are being developed to address those challenges.

A variety of technologies can be used in the detection of contagious pathogens. In the early stage of an outbreak of a new infectious disease, rtPCR is advantageous over many other assays. The rtPCR can be developed either using low fidelity DNA polymerase or high fidelity DNA polymerase. The application of high fidelity DNA polymerase allows the shortening of assay development. In addition, the synthesized DNA template used as positive controls is suggested for shortening the time for assay development. Overall comparison of time required for assay development, specificity, and sensitivity for different types of molecular diagnostic technologies, it seems that early confirmation of viral infected patients will be diagnosed primarily with PCR or rtPCR-based assays presently and likely for the near future.

Conventional biochemical assays are performed via an averaging procedure with the lysate of a large number of target cells; however, the averaged data lose information regarding the heterogeneity of individual cells. For quantitative assay of single cells, it is necessary to isolate single cells, extract cellular components and detect extremely small amounts of molecules from the individual cells. We developed new system combining a microfabricated lab-on-chip device and fluorescence correlation spectroscopy. The system features a simple protocol to isolate single-cells and detect small amounts of specific fluorescent molecules extracted from a single cell. Our results indicate that numbers of transfected DNA molecules were rather equal for each single cell; however, the expression rate of protein varied in each single cell.

p53, a tumor suppressor and a transcription factor, binds DNA in a sequence-specific manner. In more than half of human cancers, p53 has been found to be mutated with the loss of DNA-binding ability. In this review, we focus on the sensitive detection of interaction of tumor suppressor p53 with double-stranded DNA bearing the consensus sequence and proteins, such as monoclonal antibodies recognizing the p53 protein and metalloprotein. Relying on the specific binding of p53 to DNA and antibodies, quantitative determination of wild-type and mutant p53 proteins in normal and cancer cell lysates has been achieved.

This paper reviews the development of a magnetic modulation biosensing (MMB) system for rapid, simple and sensitive detection of biological targets in homogeneous solution at low concentrations. It relies on condensation and modulation of the fluorescent-labeled probes attached to magnetic beads using an alternating magnetic field gradient. Condensation of the beads from the entire volume increases the signal while modulation separates the signal from the background noise of the non-magnetized solution. We first discuss the motivation and challenges in specific DNA sequences detection as well as current approaches to overcome some of these challenges. We then present the MMB system, DNA detection schemes and magnetic beads manipulation in solution. Rapid detection at sub-picomolar concentrations of fluorescent-labeled probes as well as of coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) without any washing or separation step is also reviewed. Finally, we show preliminary results of protein detection using a ‘sandwich’-based assay.