Bacteriophage applications: where are we now?

Bacteriophages are bacterial viruses and have been used for almost a century as antimicrobial agents. In the West, their use diminished when chemical antibiotics were introduced, but they remain a common therapeutic approach in parts of eastern Europe. Increasing antibiotic resistance in bacteria has driven the demand for novel therapies to control infections and led to the replacement of antibiotics in animal husbandry. Alongside this, increased pressure to improve food safety has created a need for faster detection of pathogenic bacteria. Hence, there has been a resurgence of interest in bacteriophage applications, and this has encouraged the emergence of a large number of biotech companies hoping to commercialize their use. Research in Europe and the United States has increased steadily, leading to the development of a range of applications for bacteriophage agents for the healthcare, veterinary and agricultural sectors. This article will attempt to answer the question of whether bacteriophages are now delivering on their potential.     

细菌的应激反应和生理代谢与耐药性及其控制策略

抗菌药在医疗和畜牧生产中的滥用导致了细菌抗药性的产生,这个公共卫生问题引起了人们越来越多的关注。除了基因突变和获得形成的抗药性 (Resistance) 外,细菌在自然环境中遇到的各种压力会引发其产生应激反应,这不仅可以保护细菌免受这些压力的影响,还会改变细菌对抗菌药的耐药性 (Tolerance)。耐药性的产生必然会影响细菌的生理代谢,但是细菌可以通过调节自身代谢恢复对药物的敏感性。文中综述了近年来细菌应激反应和生理代谢与细菌耐药性之间的相关研究,以期采取更加有效的措施来控制细菌抗药性的发生和蔓延。     

Antimicrobial peptides: promising alternatives to conventional antibiotics

Antimicrobial peptides (APs) have been described as evolutionary ancient weapons. Produced by a wide variety of organisms as part of a non-specific immune response, these peptides are involved in the direct destruction of various microorganisms. Several APs have been shown to have broad activity spectra against microorganisms such as Gram-positive and Gram-negative bacteria, enveloped viruses, fungi and parasites. Given that resistance to a number of antibiotics has developed in a wide range of microbes, the potential of APs as novel therapeutic agents is being evaluated. However, optimisation of APs designed for therapy will need to focus on such factors as their susceptibility to proteolytic degradation and reduction of toxicity to mammalian cells. Strict guidelines pertaining to their use should also be established to prevent or hinder future development of bacterial resistance to such peptides.     

Developing oncolytic viruses for clinical use: A consortium approach

The use of oncolytic viruses forms an appealing approach for cancer treatment. On the one hand the viruses replicate in, and kill, tumor cells, leading to their intra-tumoral amplification. On the other hand the viral infection will activate virus-directed immune responses, and may trigger immune responses directed against tumor cells and tumor antigens. To date, a wide variety of oncolytic viruses is being developed for use in cancer treatment. While the development of oncolytic viruses has often been initiated by researchers in academia and other public institutions, a large majority of the final product development and the testing of these products in clinical trials is industry led. As a consequence relatively few pre-clinical and clinical studies evaluated different oncolytic viruses in competitive side-by-side preclinical or clinical studies. In this review we will summarize the steps and considerations essential in the development and characterization of oncolytic viruses, and describe our multidisciplinary academic consortium, which involves a dozen departments in three different Dutch universities, collaborating in the development of oncolytic viruses. This consortium has the ambition to develop a small series of oncolytic viruses and to evaluate these in various cancers.     

Adaptation of Aquatic Microbial Communities to Quaternary Ammonium Compounds

The effects of long-chain (C₁₂ to C₁₈) quaternary ammonium compounds (QACs) on the density, heterotrophic activity, and biodegradation capabilities of heterotrophic bacteria were examined in situ in a lake ecosystem. Monoalkyl and dialkyl substituted QACs were tested over a range of concentrations (0.001 to 10 mg/liter) in both acute (3 h) and chronic (21 day) exposures. In general, none of the QACs tested had significant adverse effects on bacterial densities in either acute or chronic studies. However, significant decreases in bacterial heterotrophic activity were noted in acute studies at QAC concentrations from 0.1 to 10 mg/liter. Chronic exposure of lake microbial communities to a specific monoalkyl QAC resulted in an adaptive response and recovery of heterotrophic activity. No-observable-effect level in the adapted populations was >10 mg/liter. Chronic exposure also resulted in significant increases in the number and activity of bacteria capable of biodegrading the material. The increase in biodegradation capability was observed at low (microgram per liter) concentrations which are approximately the same as realistic environmental levels. In general, our studies indicated that exposure of lake microbial communities to QACs results in the development of adapted communities which are less sensitive to potential toxic effects and more active in the biodegradation of these materials.     

Immunoprophylaxis of respiratory infections

Anti-infective antibody-based immunotherapy has gained renewed interest since the crisis of antibiotic resistance and because there is no therapy against various viral infections. The immunoprophylaxis of respiratory infections aims to utilize the ability of local antibodies to neutralize inhaled micro-organisms and their cytopathic products. Immunoglobulins for intravenous use (i.v.i.g.) have a wide spectrum of specificities. Hyperimmune i.v.i.g. containing high titers of specific antibodies have demonstrated efficacy in clinical trials, notably against the respiratory syncytial virus. Monoclonal antibodies have the advantage to be homogenous and specific for one selected epitope and several studies have demonstrated their efficacy to neutralize several infectious agents. Moreover, antibodies can be administered topically and are effective at lower doses than those needed for systemic administration. The mechanism of action could be the agglutination of bacteria or viruses at the epithelial surfaces of the respiratory tract inhibiting the early steps of the infectious process. Thanks to new technologies of humanized monoclonal antibodies, immunotherapy offers real promising perspectives for prophylactic and therapeutic therapies against a variety of current or emerging infectious diseases.     

Microbial environments confound antibiotic efficacy

The increasing prevalence of bacteria that are insensitive to our current antibiotics emphasizes the need for new antimicrobial therapies. Conventional approaches to antibacterial development that are based on the inhibition of essential processes seem to have reached the point of diminishing returns. The discovery that diverse antibiotics stimulate a common oxidative cell-death pathway represents a fundamental shift in our understanding of bactericidal antibiotic modes of action. A number of studies, as discussed above, also provide hints about how intra- and extracellular metabolism can enable antibiotic resistance and tolerance. We have, nonetheless, just begun to understand the repertoire of tactics that bacteria use to evade antibiotics. Biosynthetic pathways for natural antibiotics are ancient, and numerous mechanisms for antibiotic resistance and tolerance are likely to have evolved over the past few million years. Unraveling these mechanisms will require concerted efforts by chemical biologists, microbiologists and clinicians. These efforts will benefit from the use of metabolic models and other network-biology approaches to guide investigation of processes that modulate antibiotic susceptibility. Importantly, by helping to identify common points of vulnerability as well as key differences between pathogens, these models may lead to the development of effective adjuvants, novel antibiotics and new antimicrobial strategies. There is also a crucial need to better understand how bacteria within a population cooperate to overcome antibiotic treatments. Such investigations may benefit from the use of novel chemical probes and experimental techniques to interrogate the physiology and functional dynamics of natural microbial communities. Insights gained from these studies will augment metagenomic models that can be used to identify biomolecules responsible for these cooperative strategies. Leveraging chemical biology methodologies and systems-biology approaches for further studies of microbial environments may reveal a wealth of untapped targets for the development of novel compounds to counter the growing threat of resistant and tolerant bacterial infections.     

Current developments in microbial control of insect pests and prospects for the early 21st century

The role of microbial control in crop and forest protection and the abatement of insects of medical and veterinary importance has expanded considerably with the discovery and development of new microbial control agents and genetic improvement in bacterial and viral pathogens, and improvements in formulation, application options and compatibility with other interventions. A synopsis of the literature regarding the current use of bacteria, viruses, fungi, protozoans and nematodes as microbial control agents is presented along with speculation on their potential in the early 21st century. The most widely used of all microbial control agents isBacillus thuringiensis. The isolation within the past two decades of new strains that are larvicidal for certain Diptera and Coleoptera has increased the utility of the bacterium considerably. Further improvements in efficacy and broadening of its host range are in progress with the isolation of strains with new toxins and the manipulation ofB. thuringiensis genes that encode toxin production using both recombinant and nonrecombinant methods. Genetic manipulation of these genes has also enabled their incorporation into crop plants. The development and commercial availability of entomopathogenic nematodes in the families Steinernematidae and Heterorhabditidae expands the options for the control of insects, especially those with soil inhabiting stages. The results of natural epizootics of fungi and viruses often obviate the requirement for additional interventions. Breakthroughs in understanding the genetics ofBaculovirus and subsequent gene manipulation have increased their virulence and utility. Improved production methods that utilize insect cell culture technology may enable affordable use ofBaculovirus in the not too distant future. Fungi continue to offer the only control options using entomopathogens against plant sucking insects. Although fungi have great potential for development as microbial control agents, only a few have been used on an operational scale. Some factors that might limit the full range of entomopathogen potential, including development of resistance, are discussed. Because of their selectivity and minimal environmental impact, microbial control agents will be ideal components of integrated pest management programs in the early 21st century and beyond. However, if they are used merely as replacements for chemical pesticides, then eventually these agents will face some of the same fate as the chemicals they replace, particularly with respect to resistance.     

The role of vaccines in combating antimicrobial resistance (AMR) bacteria

Most pathogens have developed an intrinsic capacity to thrive by developing resistance to antimicrobial compounds utilized in treatment. Antimicrobial resistance arises when microbial agents such as bacteria, viruses, fungi, and parasites alter their behaviour to make current conventional medicines inefficient. Vaccination is one of the most effective strategies to fight antimicrobial resistance. Vaccines, unlike drugs, are less likely to produce resistance since they are precise to their target illnesses. Vaccines against infectious agents such as Streptococcus pneumoniae and Haemophilus influenzae have already been shown to reduce tolerance to antimicrobial medications; however, vaccines against some antimicrobial-resistant pathogens such as Vibrio cholerae, Salmonella typhi, Escherichia coli, nosocomial infections, and pulmonary and diarrheal disease viruses require more research and development. This paper describes vaccine roles in combatting antimicrobial resistance, quantifies the overall advantages of vaccination as an anti-antimicrobial resistance approach, analyzes existing antimicrobial vaccines and those currently under development, and emphasizes some of the obstacles and prospects of vaccine research and development.     

Bivalve mollusc viruses

Bivalve molluscs can bioaccumulate, in their tissues, well known viruses that infect humans and higher animals. However, since 1969 there have been reports, based largely on ultrastructural studies, that have indicated there are also viruses which infect these molluscs. Lack of bivalve molluscan cell lines has limited the isolation of these viruses, although some viruses isolated in fish cell lines are claimed to be infective for bivalves. Distinctive morphogenesis and characteristics of iridoviruses have been reported in larval and adult oysters in association with infections of epithelial and haemocytic tissues. Herpes viruses have been reported in the American oyster, Pacific oyster, and European flat oyster from three continents. The herpes viral agent in Pacific oysters has been experimentally transmitted and requires temperatures of 25–26°C for a productive infection and clinical disease. Papova-like viruses, picorna-like, and other small virus-like particles have been reported in several bivalve species. A leukemia-like disease of many bivalve species, collectively termed disseminated neoplasias, is of unconfirmed aetiology, but may be related to retroviral infections. Representatives of the Reoviridae and Birnaviridae have been isolated by using fish cell lines. One definitive study concludes that at least one example is not infective for bivalves while other studies claim molluscan infectivity. Phages have been reported in three pathogenic agents infecting bivalves. Advancement in the field of molluscan virology will require increased application of physical isolation methods, refinement of primary cell culture methods, use of molecular diagnostic tools, and the development of continuous molluscan cell lines.     

Antibody-Based Resistance to Plant Pathogens

Plant diseases are a major threat to the world food supply, as up to 15% of production is lost to pathogens. In the past, disease control and the generation of resistant plant lines protected against viral, bacterial or fungal pathogens, was achieved using conventional breeding based on crossings, mutant screenings and backcrossing. Many approaches in this field have failed or the resistance obtained has been rapidly broken by the pathogens. Recent advances in molecular biotechnology have made it possible to obtain and to modify genes that are useful for generating disease resistant crops. Several strategies, including expression of pathogen-derived sequences or anti-pathogenic agents, have been developed to engineer improved pathogen resistance in transgenic plants. Antibody-based resistance is a novel strategy for generating transgenic plants resistant to pathogens. Decades ago it was shown that polyclonal and monoclonal antibodies can neutralize viruses, bacteria and selected fungi. This approach has been improved recently by the development of recombinant antibodies (rAbs). Crop resistance can be engineered by the expression of pathogen-specific antibodies, antibody fragments or antibody fusion proteins. The advantages of this approach are that rAbs can be engineered against almost any target molecule, and it has been demonstrated that expression of functional pathogen-specific rAbs in plants confers effective pathogen protection. The efficacy of antibody-based resistance was first shown for plant viruses and its application to other plant pathogens is becoming more established. However, successful use of antibodies to generate plant pathogen resistance relies on appropriate target selection, careful antibody design, efficient antibody expression, stability and targeting to appropriate cellular compartments.     

Role of Pathogen-Related Protein 10 (PR 10) under Abiotic and Biotic Stresses in Plants

Members of the Pathogenesis Related (PR) 10 protein family have been identified in a variety of plant species and a wide range of functions ranging from defense to growth and development has been attributed to them. PR10 protein possesses ribonuclease (RNase) activity, interacts with phytohormones, involved in hormone-mediated signalling, afforded protection against various phytopathogenic fungi, bacteria, and viruses particularly in response to biotic and abiotic stresses. The resistance mechanism of PR10 protein may include activation of defense signalling pathways through possible interacting proteins involved in mediating responses to pathogens, degradation of RNA of the invading pathogens. Moreover, several morphological changes have been shown to accompany the enhanced abiotic stress tolerance. In this review, the possible mechanism of action of PR10 protein against biotic and abiotic stress has been discussed. Furthermore, our findings also confirmed that the in vivo Nitric oxide (NO) is essential for most of environmental abiotic stresses and disease resistance against pathogen infection. The proper level of NO may be necessary and beneficial, not only in plant response to the environmental abiotic stress, but also to biotic stress. The updated information on this interesting group of proteins will be useful in future research to develop multiple stress tolerance in plants.     

Microencapsulation of probiotic bacteria: technology and potential applications

In the recent past, there has been an explosion of probiotic health-based products. Many reports indicated that there is poor survival of probiotic bacteria in these products. Further, the survival of these bacteria in the human gastro-intestinal system is questionable. Providing probiotic living cells with a physical barrier against adverse environmental conditions is therefore an approach currently receiving considerable interest. The technology of micro-encapsulation of probiotic bacterial cells evolved from the immobilised cell culture technology used in the biotechnological industry. Several methods of micro-encapsulation of probiotic bacteria have been reported and include spray drying, extrusion, emulsion and phase separation. None of these reported methods however, has resulted in the large numbers of shelf-stable, viable probiotic bacterial cells necessary for use in industry for development of new probiotic products. The most commonly reported micro-encapsulation procedure is based on the calcium-alginate gel capsule formation. Kappa-carrageenan, gellan gum, gelatin and starch are also used as excipients for the micro-encapsulation of probiotic bacteria. The currently available equipment for micro-encapsulation is not able to generate large quantities of uniform sized micro or nano capsules. There is a need to design and develop equipment that will be able to generate precise and uniform micro or nano capsules in large quantities for industrial applications. The reported food vehicles for delivery of encapsulated probiotic bacteria are yoghurt, cheese, ice cream and mayonnaise. Studies need to be done on the application of micro-encapsulation of probiotic bacteria in other food systems. The number of probiotic supplements will increase in the future. More studies, however, need to be conducted on the efficacy of micro-encapsulation to deliver probiotic bacteria and their controlled or targeted release in the gastrointestinal tract.     

Eukaryotic translation initiation factor 4E-mediated recessive resistance to plant viruses and its utility in crop improvement

The use of genetic resistance is considered to be the most effective and sustainable approach to the control of plant pathogens. Although most of the known natural resistance genes are monogenic dominant R genes that are predominant against fungi and bacteria, more and more recessive resistance genes against viruses have been cloned in the last decade. Interestingly, of the 14 natural recessive resistance genes against plant viruses that have been cloned from diverse plant species thus far, 12 encode the eukaryotic translation initiation factor 4E (eIF4E) or its isoform eIF(iso)4E. This review is intended to summarize the current state of knowledge about eIF4E and the possible mechanisms underlying its essential role in virus infection, and to discuss recent progress and the potential of eIF4E as a target gene in the development of genetic resistance to viruses for crop improvement.     

Susceptibility of antibiotic-resistant cocci to biocides

Biocide resistance has hitherto been a poorly studied subject, possibly due to the belief that such resistance was rare and clinically insignificant. Various recent findings, however, have underlined the importance of biocide resistance as a clinically relevant phenomenon. Outbreaks of biocide-resistant organisms in hospitals have been described and the genetic mechanism for resistance to quaternary ammonium compounds (QACs) in Staphylococcus aureus has now been elucidated. Mycobacteria resistant to commonly used endoscope disinfectants are now commonly reported and have caused numerous adverse clinical events. Cross-resistance between triclosan and antituberculous drugs has been demonstrated in other strains of mycobacteria. This is related to a common mechanism of action. The work presented here describes studies into the biocide resistance of antibiotic-resistant cocci and attempts to create biocide-resistant strains in vitro. Strains of staphylococci (including methicillin-resistant Staph. aureus (MRSA)) and enterococci (including vancomycin-resistant enterococci (VRE)) had their susceptibility to biocides assayed using broth macro dilution methods and resistant strains were selected by serial subculture on biocide-containing media. Mutants were created with relative ease; for instance, triclosan minimal bactericidal concentrations (MBCs) increased from 0.002 to 3.12 mg l(-1). Some strains of MRSA which have intermediate resistance to glycopeptides were demonstrated to have decreased susceptibility to some biocides. Biocide resistance amongst enterococci was demonstrated although there was no clear correlation between biocide and antibiotic resistance. The exact mechanisms of resistance in these strains are still being studied but it is clear that biocide resistance is an important clinical phenomenon.     

Elm defence against herbivores and pathogens: morphological,chemical and molecular regulation aspects

Elms (Ulmus spp.) have long been appreciated for their environmental tolerance, landscape and ornamental value, and the quality of their wood. Although elm trees are extremely hardy against abiotic stresses such as wind and pollution, they are susceptible to attacks of biotic stressors. Over 100 phytopathogens and invertebrate pests are associated with elms: fungi, bacteria and insects like beetles and moths, and to a lesser extent aphids, mites, viruses and nematodes. While the biology of the pathogen and insect vector of the Dutch elm disease has been intensively studied, less attention has been paid so far to the defence mechanisms of elms to other biotic stressors. This review highlights knowledge of direct and indirect elm defences against biotic stressors focusing on morphological, chemical and gene regulation aspects. First, we report how morphological defence mechanisms via barrier formation and vessel occlusion prevent colonisation and spread of wood- and bark-inhabiting fungi and bacteria. Second, we outline how secondary metabolites such as terpenoids (volatile terpenoids, mansonones and triterpenoids) and phenolics (lignans, coumarins, flavonoids) in leaves and bark are involved in constitutive and induced chemical defence mechanisms of elms. Third, we address knowledge on how the molecular regulation of elm defence is orchestrated through the interaction of a huge variety of stress- and defence-related genes. We conclude by pointing to the gaps of knowledge on the chemical and molecular mechanisms of elm defence against pest insects and diseases. An in-depth understanding of defence mechanisms of elms will support the development of sustainable integrated management of pests and diseases attacking elms.     

Vaspin (serpinA12) in obesity,insulin resistance,and inflammation

While genome‐wide association studies as well as candidate gene studies have revealed a great deal of insight into the contribution of genetics to obesity development and susceptibility, advances in adipose tissue research have substantially changed the understanding of adipose tissue function. Its perception has changed from passive lipid storage tissue to active endocrine organ regulating and modulating whole‐body energy homeostasis and metabolism and inflammatory and immune responses by secreting a multitude of bioactive molecules, termed adipokines. The expression of human vaspin (serpinA12) is positively correlated to body mass index and insulin sensitivity and increases glucose tolerance in vivo, suggesting a compensatory role in response to diminished insulin signaling in obesity. Recently, considerable insight has been gained into vaspin structure, function, and specific target tissue‐dependent effects, and several lines of evidence suggest vaspin as a promising candidate for drug development for the treatment of obesity‐related insulin resistance and inflammation. These will be summarized in this review with a focus on molecular mechanisms and pathways. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Harnessed viruses in the age of metagenomics and synthetic biology: an update on infectious clone assembly and biotechnologies of plant viruses

Recent metagenomic studies have provided an unprecedented wealth of data, which are revolutionizing our understanding of virus diversity. A redrawn landscape highlights viruses as active players in the phytobiome, and surveys have uncovered their positive roles in environmental stress tolerance of plants. Viral infectious clones are key tools for functional characterization of known and newly identified viruses. Knowledge of viruses and their components has been instrumental for the development of modern plant molecular biology and biotechnology. In this review, we provide extensive guidelines built on current synthetic biology advances that streamline infectious clone assembly, thus lessening a major technical constraint of plant virology. The focus is on generation of infectious clones in binary T‐DNA vectors, which are delivered efficiently to plants by Agrobacterium. We then summarize recent applications of plant viruses and explore emerging trends in microbiology, bacterial and human virology that, once translated to plant virology, could lead to the development of virus‐based gene therapies for ad hoc engineering of plant traits. The systematic characterization of plant virus roles in the phytobiome and next‐generation virus‐based tools will be indispensable landmarks in the synthetic biology roadmap to better crops.     

Resistance to antimicrobial peptides in Gram-negative bacteria

Antimicrobial peptides (AMPs) are present in virtually all organisms and are an ancient and critical component of innate immunity. In mammals, AMPs are present in phagocytic cells, on body surfaces such as skin and mucosa, and in secretions and body fluids such as sweat, saliva, urine, and breast milk, consistent with their role as part of the first line of defense against a wide range of pathogenic microorganisms including bacteria, viruses, and fungi. AMPs are microbicidal and have also been shown to act as immunomodulators with chemoattractant and signaling activities. During the co-evolution of hosts and bacterial pathogens, bacteria have developed the ability to sense and initiate an adaptive response to AMPs to resist their bactericidal activity. Here, we review the various mechanisms used by Gram-negative bacteria to sense and resist AMP-mediated killing. These mechanisms play an important role in bacterial resistance to host-derived AMPs that are encountered during the course of infection. Bacterial resistance to AMPs should also be taken into consideration in the development and use of AMPs as anti-infective agents, for which there is currently a great deal of academic and commercial interest.