Abundance of Antibiotic Resistance Genes in Bacteriophage following Soil Fertilization with Dairy Manure or Municipal Biosolids,and Evidence for Potential Transduction

Animal manures and municipal biosolids recycled onto crop production land carry antibiotic-resistant bacteria that can influence the antibiotic resistome of agricultural soils, but little is known about the contribution of bacteriophage to the dissemination of antibiotic resistance genes (ARGs) in this context. In this work, we quantified a set of ARGs in the bacterial and bacteriophage fractions of agricultural soil by quantitative PCR. All tested ARGs were present in both the bacterial and phage fractions. We demonstrate that fertilization of soil with dairy manure or human biosolids increases ARG abundance in the bacterial fraction but not the bacteriophage fraction and further show that pretreatment of dairy manure can impact ARG abundance in the bacterial fraction. Finally, we show that purified bacteriophage can confer increased antibiotic resistance to soil bacteria when combined with selective pressure. The results indicate that soilborne bacteriophage represents a substantial reservoir of antibiotic resistance and that bacteriophage could play a significant role in the horizontal transfer of resistance genes in the context of an agricultural soil microbiome. Overall, our work reinforces the advisability of composting or digesting fecal material prior to field application and suggests that application of some antibiotics at subclinical concentrations can promote bacteriophage-mediated horizontal transfer of ARGs in agricultural soil microbiomes.     

Enhancement of the antimicrobial properties of bacteriophage‐K via stabilization using oil‐in‐water nano‐emulsions

Bacteriophage therapy is a promising new treatment that may help overcome the threat posed by antibiotic‐resistant pathogenic bacteria, which are increasingly identified in hospitalized patients. The development of biocompatible and sustainable vehicles for incorporation of viable bacterial viruses into a wound dressing is a promising alternative. This article evaluates the antimicrobial efficacy of Bacteriophage K against Staphylococcus aureus over time, when stabilized and delivered via an oil‐in‐water nano‐emulsion. Nano‐emulsions were formulated via thermal phase inversion emulsification, and then bacterial growth was challenged with either native emulsion, or emulsion combined with Bacteriophage K. Bacteriophage infectivity, and the influence of storage time of the preparation, were assessed by turbidity measurements of bacterial samples. Newly prepared Bacteriophage K/nano‐emulsion formulations have greater antimicrobial activity than freely suspended bacteriophage. The phage‐loaded emulsions caused rapid and complete bacterial death of three different strains of S. aureus. The same effect was observed for preparations that were either stored at room temperature (18–20°C), or chilled at 4°C, for up to 10 days of storage. A response surface design of experiments was used to gain insight on the relative effects of the emulsion formulation on bacterial growth and phage lytic activity. More diluted emulsions had a less significant effect on bacterial growth, and diluted bacteriophage‐emulsion preparations yielded greater antibacterial activity. The enhancement of bacteriophage activity when delivered via nano‐emulsions is yet to be reported. This prompts further investigation into the use of these formulations for the development of novel anti‐microbial wound management strategies. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:932–944, 2014     

Bacteriophage lytic enzymes: novel anti-infectives

Bacteriophage lytic enzymes, or lysins, are highly evolved molecules produced by bacterial viruses (bacteriophage) to digest the bacterial cell wall for bacteriophage progeny release. Small quantities of purified recombinant lysin added to gram-positive bacteria causes immediate lysis resulting in log-fold death of the target bacterium. Lysins have now been used successfully in animal models to control pathogenic antibiotic resistant bacteria found on mucosal surfaces and in blood. The advantages over antibiotics are their specificity for the pathogen without disturbing the normal flora, the low chance of bacterial resistance to lysins and their ability to kill colonizing pathogens on mucosal surfaces, capabilities that were previously unavailable. Thus, lysins could be an effective anti-infective in an age of mounting antibiotic resistance.     

Listeria monocytogenes wall teichoic acid decoration in virulence and cell‐to‐cell spread

Wall teichoic acid (WTA) comprises a class of glycopolymers covalently attached to the peptidoglycan of gram positive bacteria. In Listeria monocytogenes, mutations that prevent addition of certain WTA decorating sugars are attenuating. However, the steps required for decoration and the pathogenic process interrupted are not well described. We systematically examined the requirement for WTA galactosylation in a mouse oral‐virulent strain by first creating mutations in four genes whose products conferred resistance to a WTA‐binding bacteriophage. WTA biochemical and structural studies indicated that galactosylated WTA was directly required for bacteriophage adsorption and that mutant WTA lacked appreciable galactose in all except one mutant – which retained a level ca. 7% of the parent. All mutants were profoundly attenuated in orally infected mice and were impaired in cell‐to‐cell spread in vitro. Confocal microscopy of cytosolic mutants revealed that all expressed ActA on their cell surface and formed actin tails with a frequency similar to the parent. However, the mutant tails were significantly shorter – suggesting a defect in actin based motility. Roles for the gene products in WTA galactosylation are proposed. Identification and interruption of WTA decoration pathways may provide a general strategy to discover non‐antibiotic therapeutics for gram positive infections. © 2016 John Wiley & Sons Ltd     

Bacteriophages and bacteriophage‐derived endolysins as potential therapeutics to combat Gram‐positive spore forming bacteria

Since their discovery in 1915, bacteriophages have been routinely used within Eastern Europe to treat a variety of bacterial infections. Although initially ignored by the West due to the success of antibiotics, increasing levels and diversity of antibiotic resistance is driving a renaissance for bacteriophage‐derived therapy, which is in part due to the highly specific nature of bacteriophages as well as their relative abundance. This review focuses on the bacteriophages and derived lysins of relevant Gram‐positive spore formers within the Bacillus cereus group and Clostridium genus that could have applications within the medical, food and environmental sectors.     

Evaluation of lytic activity of staphylococcal bacteriophage Sb‐1 against freshly isolated clinical pathogens

In recent decades the increase in antibiotic‐resistant bacterial strains has become a serious threat to the treatment of infectious diseases. Drug resistance of Staphylococcus aureus has become a major problem in hospitals of many countries, including developed ones. Today the interest in alternative remedies to antibiotics, including bacteriophage treatment, is gaining new ground. Here, we describe the staphylococcal bacteriophage Sb‐1 – a key component of therapeutic phage preparation that was successfully used against staphylococcal infections during many years in the Former Soviet Union. This phage still reveals a high spectrum of lytic activity in vitro against freshly isolated, genetically different clinical samples (including methicillin‐resistant S. aureus) obtained from the local hospitals, as well as the clinics from different geographical areas. The sequence analyses of phage genome showed absence of bacterial virulence genes. A case report describes a promising clinical response after phage application in patient with cystic fibrosis and indicates the efficacy of usage of Sb‐1 phage against various staphylococcal infections.     

Use of bacteriophage endolysin EL188 and outer membrane permeabilizers against Pseudomonas aeruginosa

Aims: To select and evaluate an appropriate outer membrane (OM) permeabilizer to use in combination with the highly muralytic bacteriophage endolysin EL188 to inactivate (multi‐resistant) Pseudomonas aeruginosa. Methods and Results: We tested the combination of endolysin EL188 and several OM permeabilizing compounds on three selected Ps. aeruginosa strains with varying antibiotic resistance. We analysed OM permeabilization using the hydrophobic probe N‐phenylnaphtylamine and a recombinant fusion protein of a peptidoglycan binding domain and green fluorescent protein on the one hand and cell lysis assays on the other hand. Antibacterial assays showed that incubation of 10⁶Ps. aeruginosa cells ml^?1 in presence of 10 mmol l^?1 ethylene diamine tetraacetic acid disodium salt dihydrate (EDTA) and 50 μg ml^?1 endolysin EL188 led to a strain‐dependent inactivation between 3·01 ± 0·17 and 4·27 ± 0·11 log units in 30 min. Increasing the EL188 concentration to 250 μg ml^?1 further increased the inactivation of the most antibiotic resistant strain Br667 (4·07 ± 0·09 log units). Conclusions: Ethylene diamine tetraacetic acid disodium salt dihydrate was selected as the most suitable component to combine with EL188 in order to reduce Ps. aeruginosa with up to 4 log units in a time interval of 30 min. Significance and Impact of the Study: This in vitro study demonstrates that the application range of bacteriophage encoded endolysins as ‘enzybiotics’ must not be limited to gram‐positive pathogens.     

Structural changes induced by a lytic bacteriophage make ciprofloxacin effective against older biofilm of Klebsiella pneumoniae

Bacteria have evolved multiple mechanisms, such as biofilm formation, to thwart antibiotic action. Yet antibiotics remain the drug of choice against clinical infections. It has been documented that young biofilm of Klebsiella pneumoniae could be eradicated significantly by ciprofloxacin treatment alone. Since age of biofilm is a decisive factor in determining the outcome of antibiotic treatment, in the present study biofilm of K. pneumoniae, grown for extended periods was treated with ciprofloxacin and/or depolymerase producing lytic bacteriophage (KPO1K2). The reduction in bacterial numbers of older biofilm was greater after application of the two agents in combination as ciprofloxacin alone could not reduce bacterial biomass significantly in older biofilms (P > 0.05). Confocal microscopy suggested the induction of structural changes in the biofilm matrix and a decrease in micro-colony size after KPO1K2 treatment. The role of phage associated depolymerase was emphasized by the insignificant eradication of biofilm by a non-depolymerase producing bacteriophage that, however, eradicated the biofilm when applied concomitantly with purified depolymerase. These findings demonstrate that a lytic bacteriophage alone can eradicate older biofilms significantly and its action is primarily depolymerase mediated. However, application of phage and antibiotic in combination resulted in slightly increased biofilm eradication confirming the speculation that antibiotic efficacy can be augmented by bacteriophage.     

Isolation and partial characterization of Salmonella Gallinarum bacteriophage

Infections caused by Salmonella remain a major public health problem worldwide. Animal food products, including poultry meat and eggs, are considered essential components in the individual’s daily nutrition. However, chicken continues to be the main reservoir for Salmonella spp.Poultry farmers use several types of antibiotics to treat pathogens. This can pose a health risk as pathogens can build antibiotic resistance in addition to the possibility of accumulation of these antibiotics in food products. The use of phages in treating poultry pathogens is increasing worldwide due to its potential use as an effective alternative to antibiotics. Phages have several advantages over antibiotics; phages are very specific to target bacteria, less chances of developing secondary infections, and they only replicate at the site of infection.Here we report the isolation of a bacteriophage from chicken feces. The isolated bacteriophage hosts on Salmonella Gallinarum, a common zoonotic infection that causes fowl typhoid, known to cause major losses to poultry sector. The isolated bacteriophage was partially characterized as a DNA virus resistant to RNase digestion with approximately 20 Kb genome. SDS-PAGE analysis of total viral proteins showed at least five major bands (21, 28, 42, 55 and 68 kDa), indicating that this virus is relatively small compared to other known poultry phages. The isolated bacteriophage has the potential to be an alternative to antibiotics and possibly reducing antibiotic resistance in poultry farms.     

噬菌体裂解酶——现状与未来

噬菌体裂解酶是一种由DNA噬菌体基因编码的高特异性酶, 可高效消化细菌细胞壁。革兰氏阳性菌噬菌体裂解酶的结构域相似, 裂解效率高, 与抗生素具协同抗菌作用, 且不易产生耐受性菌株, 抗体等体液因子对裂解酶的裂解活性影响小, 裂解酶作为一种潜在抗感染药物具有重要的研究价值。目前已建立了多种病原菌裂解酶应用的动物模型, 在防控耐药性病原菌感染上取得重要进展。本文就噬菌体裂解酶的抗菌作用进行综述。     

A Window of Opportunity to Control the Bacterial Pathogen Pseudomonas aeruginosa Combining Antibiotics and Phages

The evolution of antibiotic resistance in bacteria is a global concern and the use of bacteriophages alone or in combined therapies is attracting increasing attention as an alternative. Evolutionary theory predicts that the probability of bacterial resistance to both phages and antibiotics will be lower than to either separately, due for example to fitness costs or to trade-offs between phage resistance mechanisms and bacterial growth. In this study, we assess the population impacts of either individual or combined treatments of a bacteriophage and streptomycin on the nosocomial pathogen Pseudomonas aeruginosa. We show that combining phage and antibiotics substantially increases bacterial control compared to either separately, and that there is a specific time delay in antibiotic introduction independent of antibiotic dose, that minimizes both bacterial density and resistance to either antibiotics or phage. These results have implications for optimal combined therapeutic approaches.     

Biological control of microbial infections and cancer in humans: Historical use to future potential

Prior to the advent of antibiotics, live organisms were used directly in attempts to control microbial infections and cure cancers. Examples of such biological control included bacteriotherapy, bacteriophage therapy, malaria therapy, probiotics and the use of living maggots. In all cases, the organisms themselves, rather than products of their metabolism, were used as the potentially curative agents. The history of the use of biocontrol agents in the treatment of human infections and cancer is discussed here in relation to more recent examples of the use of this approach. Modern studies suggest that the use of biological control in the treatment of human infections may be worth re‐evaluating in the light of the increasing world‐wide occurrence of antibiotic‐resistant bacteria and the opportunities provided by recent developments in gene technology.     

Development and validation of a qPCR‐based method for quantifying Shiga toxin‐encoding and other lambdoid bacteriophages

To address whether seasonal variability exists among Shiga toxin‐encoding bacteriophage (Stx phage) numbers on a cattle farm, conventional plaque assay was performed on water samples collected over a 17 month period. Distinct seasonal variation in bacteriophage numbers was evident, peaking between June and August. Removal of cattle from the pasture precipitated a reduction in bacteriophage numbers, and during the winter months, no bacteriophage infecting Escherichia coli were detected, a surprising occurrence considering that 10³¹ tailed‐bacteriophages are estimated to populate the globe. To address this discrepancy a culture‐independent method based on quantitative PCR was developed. Primers targeting the Q gene and stx genes were designed that accurately and discriminately quantified artificial mixed lambdoid bacteriophage populations. Application of these primer sets to water samples possessing no detectable phages by plaque assay, demonstrated that the number of lambdoid bacteriophage ranged from 4.7 × 10⁴ to 6.5 × 10⁶ ml^?1, with one in 10³ free lambdoid bacteriophages carrying a Shiga toxin operon (stx). Specific molecular biological tools and discriminatory gene targets have enabled virus populations in the natural environment to be enumerated and similar strategies could replace existing propagation‐dependent techniques, which grossly underestimate the abundance of viral entities.     

Saturated Fatty Acid Mutant of Saccharomyces cerevisiae with an Intact Fatty Acid Synthetase

To determine directly the effects of streptomycin on translational fidelity in intact cells, we studied the synthesis of beta-galactosidase and of the coat protein of bacteriophage R17 in an Escherichia coli mutant in which the bactericidal effects of streptomycin are delayed. After the addition of streptomycin to exponentially growing mutant cells, protein synthesis continues at an undiminished rate for approximately an hour; however, as measured by enzyme assays, little functional protein is produced. Serological assays designed to detect beta-galactosidase and bacteriophage R17 coat protein show that substantial amounts of the protein synthesized can react with antisera prepared against active beta-galactosidase and phage R17, indicating the aberrance of the protein produced in the presence of the antibiotic. The polypeptides synthesized in the presence of streptomycin are degraded in the cell to a much greater extent than protein synthesized in the absence of the antibiotic. The proteolytic attack on this protein is not affected by inhibitors of serine proteases, suggesting that enzymes other than those involved in "normal turnover" of cellular protein are responsible. In this strain, certain of the multiple effects of streptomycin are separated in time and the production of abnormal protein (enzymatically inactive and susceptible to proteolytic attack) could be studied in the absence of the lethal effect of the drug.     

Incorporation of T4 bacteriophage in electrospun fibres

Aims

Antibacterial food packaging materials, such as bacteriophage‐activated electrospun fibrous mats, may address concerns triggered by waves of bacterial food contamination. To address this, we investigated several efficient methods for incorporating T4 bacteriophage into electrospun fibrous mats.

Methods and Results

The incorporation of T4 bacteriophage using simple suspension electrospinning led to more than five orders of magnitude decrease in bacteriophage activity. To better maintain bacteriophage viability, emulsion electrospinning was developed where the T4 bacteriophage was pre‐encapsulated in an alginate reservoir via an emulsification process and subsequently electrospun into fibres. This resulted in an increase in bacteriophage viability, but there was still two orders of magnitude drop in activity. Using a coaxial electrospinning process, full bacteriophage activity could be maintained. In this process, a core/shell fibre structure was formed with the T4 bacteriophage being directly incorporated into the fibre core. The core/shell fibre encapsulated bacteriophage exhibited full bacteriophage viability after storing for several weeks at +4°C.

Conclusions

Coaxial electrospinning was shown to be capable of encapsulating bacteriophages with high loading capacity, high viability and long storage time.

Significance and Impact of the Study

These results are significant in the context of controlling and preventing bacterial infections in perishable foods during storage.     

Application of Paper Chromatograms to the Study of Inducers of λ Bacteriophage in Escherichia coli

A procedure has been developed whereby paper chromatograms of agents which induce lambda bacteriophage in Escherichia coli can be developed using bioautographs with a lysogenic test system. Well-defined plaque-forming zones are produced indicating the area on the paper chromatogram where the active inducing material can be located. A mixture of the bacteriophage-inducing antibiotic, mitomycin C, and the noninducing antibiotic, paromomycin, was resolved into its components on paper strips with an ethyl acetate-methanol solvent system. The location of both antibiotics could thus be readily observed. Antibacterial and inducing activities were found to be identical with a crude fermentation solid, NSC-B-158,791. The use of this procedure for resolution of multicomponent inducing activities in antibiotic beers and for characterization of active components which may be potential antitumor antibiotics is indicated.     

Synergistic phage-antibiotic combinations for the control of Escherichia coli biofilms in vitro

The potential application of phage therapy for the control of bacterial biofilms has received increasing attention as resistance to conventional antibiotic agents continues to increase. The present study identifies antimicrobial synergy between bacteriophage T4 and a conventional antibiotic, cefotaxime, via standard plaque assay and, importantly, in the in vitro eradication of biofilms of the T4 host strain Escherichia coli 11303. Phage-antibiotic synergy (PAS) is defined as the phenomenon whereby sub-lethal concentrations of certain antibiotics can substantially stimulate the host bacteria's production of virulent phage. Increasing sub-lethal concentrations of cefotaxime resulted in an observed increase in T4 plaque size and T4 concentration. The application of PAS to the T4 one-step growth curve also resulted in an increased burst size and reduced latent period. Combinations of T4 bacteriophage and cefotaxime significantly enhanced the eradication of bacterial biofilms when compared to treatment with cefotaxime alone. The addition of medium (10(4) PFU mL(-1)) and high (10(7) PFU mL(-1)) phage titres reduced the minimum biofilm eradication concentration value of cefotaxime against E. coli ATCC 11303 biofilms from 256 to 128 and 32 μg mL(-1), respectively. Although further investigation is needed to confirm PAS, this study demonstrates, for the first time, that synergy between bacteriophage and conventional antibiotics can significantly improve biofilm control in vitro.