When virus hides as part of bacterial chromosome

PCR usually cannot differentiate between viable and non-viable cells unless the sample to be tested is treated with propidium monoazide [ 70 , 71 ]. Due to the limitation of these techniques, there is a need for novel technology to be rapid, specific, reliable and easy to use. Biosensors present an intelligent alternative to the systems outlined above. A biosensor is a detection system that converts a biological response into a measurable signal. A biosensor consists of four main elements, a bio-recognition element, a signal transduction platform, a signal amplifier and a signal display [ 69 ].

The variety of biosensors available and benefits of these systems will be discussed in a later section. The specific phage—host interactions discussed above may be exploited, particularly for the detection of foodborne pathogens. Due to the issues with more traditional detection methods outlined above, recent research has now been focused on the exploitation of phage—host interactions for the detection of foodborne pathogens [ 72 ].

Detection systems based on phage—host interactions do not have the same incapacity as PCR-based systems; as they will detect only living cells these systems can obtain results more rapidly than culture-based techniques and do not require highly trained personnel. The application of whole phages, phage-derived proteins and biosensors for this purpose will now be discussed. The simplest and most direct method to detect foodborne pathogens using whole phages is the generation and enumeration of plaques on a lawn of bacteria in a method known as the phage amplification assay [ 73 ].

The sample to be tested is combined with phages that are specific for the pathogen in question, and phages are given time to adsorb and bind to their host cells. If the titer of the phages increases, it correlates to successful binding of the phage to the host cell, leading to lysis and release of progeny phages and thus the presence of the viable target in the food sample if indicated.

This type of method was employed by Jung and Ahn [ 74 ] for the detection of Shigella boydii in artificially inoculated lettuce and chicken breast and in pure culture.

The assay resulted in the detection of Shigella boydii in both single and mixed cultures E. No significant difference was noted between single and mixed cultures in the enumeration of plaques which indicates that this assay can specifically identify Shigella boydii. However, differences between the number of colonies and plaques were consistently noted in lettuce 6.

The team suggest that this may be due to the adsorption rate of the bacteriophages and the hindrance of the food matrix [ 74 ]. Enteritidis in spiked chicken meat samples. A total of 0. Enteritidis could be detected within 10 h. It was also shown that if the concentration of S. The time required to obtain results is an issue when using phage to detect Bacillus anthracis B. When using LFI lateral flow immunochromatography devices to assay phage amplification as a method of bacterial detection it is based on the detection of progeny phages as opposed to the input of phage to initiate infection.

Following 2 h a positive result was obtained for an input of B. Although this LOD seems quite high the team noted that this is a significant improvement over culture-based detection methods which require 12— h to obtain the same result [ 76 ]. Another area that focuses on whole-phage detection systems is the use of recombinantly engineered phages reporter phages.

The mechanism of action of these reporter phage systems is based on the modification of phage genomes to incorporate a bioluminescence or fluorescence gene that the phage alone cannot express. Similarly, reporter phage systems can be created to allow detection based on enzymatic conversion of a chromogenic substrate.

The genome of the E. Modification of the phage to express NanoLuc luciferase Nluc allowed bioluminescent-based detection of E. This assay detected 5. When testing for E. Both results required a pre-enrichment step in sample preparation. This technique was also adapted for the detection of S.

Typhimurium was detected using this bioluminescent reporter phage within 2 h. Results from this experiment also showed that bioluminescent signals increased proportionally to the number of cells present in lettuce, milk and sliced pork, indicating that the reporter phage successfully detects live S. Typhimurium [ 78 ]. Fluorescent reporter phages usually have integrated fluorescent molecules such as GFP in the genome of the phage so that these phages may be applied to detection assays.

The GFP gene is relatively small approx. The advantages of using GFP include its stability and autofluorescence which means there is no requirement of a substrate for activation. When L. This particular assay had a detection limit for L. This research also showed the practicality of these types of assays, when chocolate milk and salmon were spiked with L.

Phage proteins that are responsible for the adsorption of the phage to a specific host cell, such as RBPS and CBDs, may also be integrated into systems for the detection of foodborne pathogens. The genome of Campylobacter jejuni C. This protein was applied to a simple glass slide agglutination assay for the detection of C. Assays such as this can be performed in minutes and are very cost-effective in comparison to other detection systems available [ 81 ]. Phage-derived proteins were also exploited by Denyes [ 82 ] and team for the detection of Salmonella cells whereby the binding specificity of the LTFs of S16 was harnessed as an affinity molecule.

Complexes of recombinant gp37—gp38 LTF were coated onto paramagnetic beads MBs for the magnetic separation and enrichment of Salmonella. The recovery efficiency of the LTF—MBs was tested on pre-enriched food samples chicken, infant formula, milk and chocolate milk. Plating of the bead-captured Salmonella resulted in highly sensitive detection of S. Typhimurium, however, the technique is not rapid, and the integration of the LTF-based enrichment into a sandwich assay with horseradish-peroxidase HRP was investigated to overcome the issue of time.

Typhimurium cells at a concentration of 10 2 were detected in 2 h using this assay [ 82 ]. Using phage tailed proteins in conjunction with solid phase support SPS to simply and rapidly detect foodborne pathogens E.

Here the team functionalized the surface of SPS with specific phage tail proteins to target the pathogen of interest. This SPS is placed into the primary food enrichment bag after stomaching. The sample is incubated for the required time and following this the captured bacteria are detected visually in situ due to the bacterial reduction of the colourless soluble substrate triphenyltetrazolium chloride TTC to an insoluble formazan product intracellular red.

When testing foods contaminated with E. The SPS was also functionalized using specific Listeria spp. Following 40 h of incubation, positive results were obtained for both strains.

Salmonella Napoli and S. Typhimurium were artificially inoculated in egg, pastry and ground beef and gave positive results. When testing S. Typhimurium in eggs, a pale red positive result was obtained [ 83 ]. The studies above indicate that phage-derived proteins may be applied for the rapid and sensitive detection of foodborne pathogens. The utilization of phages to act as biorecognition elements in a biosensor is an established idea, to allow rapid, specific and highly sensitive detection of the bacteria in question.

Biosensing systems are composed of a recognition element, a sensor surface, a transduction platform, an amplifier, a detector and a signal output. The sensitivity and specificity of the overall system depends on the transduction signal employed and what bio-probe is used.

Figure 6 outlines the mandatory components of a biosensor. The majority of systems that utilize phages as recognition elements use the whole phages immobilized onto a solid substrate such as the phage M13 immobilised on a gold surface for detection of Salmonella spp.

The components of a biosensor, displaying the various recognition elements that may be attached to the sensors surface.

Along with the use of whole phages, many of the initial sensing systems use surface plasmon resistance SPR biosensors based on optical transduction. SPR is a phenomenon that occurs when a beam of polarized light hits a metal surface at the interface of media with a different refractive index.

Sensing techniques that use SPR are based on the principle that under specific conditions surface plasmons on the surface of a metal film may be excited by photons and transform a photon into a surface plasmon depending on the refractive index of the adsorbate.

The most common geometrical set-up of SPR is the Kretschmann configuration. The incoming polarized light hits the metallic film on the opposite side of where the adsorbate is located. The photons induce an evanescent field into the metallic film. Whenever a plasmon is excited, one photon disappears producing a dip in reflected light at that specific angle.

The angle which is dependent on the refractive index of that adsorbate is measured with a charged couple device chip. When the molecule to be detected has absorbed to the surface, the difference between the refractive index of the buffer and refractive index of the molecule can be converted into mass and thickness of the target molecule. In a study carried out by Balasubramanian and team, a biosensor was created to detect S.

In this experiment, the whole phages were immobilized onto the gold surface of a SPREETA sensor via direct physical adsorption, avoiding complex surface chemistry and phage modification. Although this system is a simple to use alternative to label-based systems, SPR based systems have been criticized due to their high LOD 1.

Systems with a lower detection limit would be more applicable to the food testing industry. Niyomdecha et al. The mechanism of action of a capacitive measurement is based on the electrical double layer on the surface of a metal electrode.

An electrode is fixed with a biosensing element and has a stable capacitance response. Binding of the target bacteria to the biosensing element of the surface results in a decrease in capacitance. This device was capable of detecting 2. In order to improve the limit of detection in this system, the team suggested using a lower flow rate or a higher sample volume, both of which would provide a longer contact time between the cell and the analytes.

A consequence of this is a longer response time. Although the two systems just described resulted in the accurate detection of foodborne pathogens in a rapid and sensitive manner, the issues associated with using whole phages as the recognition elements cannot be ignored. The incorrect orientation of bacteriophage on the surface of the platform may play a role in the sensitivity of the device.

Obtaining the correct orientation of the phages is one major issue that must be overcome by the manufacturers of the device. In addition, it has also been reported that phages lose their activity during drying following fixation on a surface [ 88 ].

The hypothesis here being that the phage heads would attach to a positively charged surface, leaving the tail fibers available to capture the bacteria— E. The cocktail of bound phage onto a positively charged cellulose membrane was examined to control the growth of L. Research is now primarily focused on using phage-derived proteins for detection of the host cells. Since CBDs have a strong affinity and specificity for the target bacteria and can be easily cloned in an E.

Singh et al. In this study, the gp48 protein was expressed as a fusion protein with a glutathione S-transferase GST tag to aid in its purification. The results also showed that the addition of a GST tag prior to immobilization of gp48 provided optimal orientation on the surface which improves the subsequent host capture in comparison to techniques based on random orientation.

The binding experiments were performed using pure cultures of C. For almost a century, phages have been used as antimicrobial agents. In the Western hemisphere, the use of phages for this purpose drastically diminished with the emergence of chemical antibiotics, however, they are still heavily used as therapeutics in parts of Eastern Europe. With the emergence of antibiotic resistance, the identification of novel antimicrobials to combat these resistant strains is more important than ever.

The ability of multi-drug resistant MDR bacteria to enter the food chain during slaughtering requires efforts to be made for the elimination of these bacteria.

MDR bacteria can enter the food chain from the environment via the contamination of ground surface water or through spraying of food crops with water containing MDR bacteria derived from animal and human waste [ 91 ].

The use of phages to target these resistant bacterial strains is a promising area of research. Ideally, the candidate phage should have a broad host range and also be exclusively virulent to avoid the risk of transmission of bacterial DNA by transduction.

While the use of single phages for detection of a foodborne pathogen is useful due to their specificity, as a biocontrol agent or tool, the use of single phages is ineffective due to their limited host range and the host cell may have systems in place to create resistance to this phage blocking of phage receptors, production of extracellular matrix, production of competitive inhibitors, preventing phage DNA entry, slicing phage nucleic acids and abortive infection mechanisms [ 18 ].

To negate this issue, a number of options have been investigated including the use of phage cocktails, containing mixtures of phages specific for the target pathogen. Other phage-based food processing aids that have been approved include Salmonelex cocktail of phages produced by Micreos Food Safety, and EcoShield cocktail of phages and SalmoFresh cocktail of six phages from Intralytix. Typhimurium in RTE foods spiked with this bacterium [ 93 ]. Typhimurium cells were recovered.

Although broad host range phages exist for a variety of bacteria, it can be difficult to isolate broad host range phages for a specific bacterium of interest. Therefore, when phages are investigated as a biocontrol agent in research settings, often it is a combination of phages with narrow and varying host ranges, known as a phage cocktail that is employed.

There are many antiviral mechanisms that may be employed by the host cell to evade infection from phages which have resulted in the emergence of phage-insensitive bacteria strains [ 18 ].

Phage cocktails containing phages that target different receptors of a host cell may reduce the colonisation of foodborne pathogens in foods without the development of phage-insensitive bacteria [ 94 ].

The use of phage cocktails, as opposed to single phages, can moderate and delay the emergence of phage resistance, as demonstrated by Fischer et al. In this example, the authors compared the application of a single phage and a four-phage cocktail in broilers on reduction of C. The percentage of isolates demonstrating resistance to each of the four individual phages ranged from In contract, the percentage of isolates demonstrating resistance to the cocktail was 0. Bai et al.

Typhimurium in fresh produce using phages that target different receptors of S. Fresh lettuce and cucumber were spiked with S. Treatment with the phage cocktail resulted in a 4.

Research such as this highlights the potential of using phage cocktails as an antimicrobial in RTE food products. Similarly, Coffey et al. Results showed that following an hour after treatment there was no significant reduction in E.

The genomes of phages can now be easily sequenced and analysed and the information exploited to identify phage-derived proteins which may themselves be used as biocontrol agents to prevent outbreaks of foodborne illness. With the addition of the protein to soya milk spiked with L. Optimal activity was seen at room temperature, with the protein reducing S.

Pre-harvest and post-harvest treatment of food-producing plants is an area of interest to both the industry and researchers due to the economic loss and the threat of illness caused by foodborne pathogens. Das et al. In this set of experiments the prophylactic treatment was deemed successful with vines which were not treated with phage displaying symptoms of PD and vines treated with the phage cocktail not displaying any symptoms over the course of the experiment [ ].

The effectiveness of Listex P as a post-harvest treatment to control the occurrence of L. Fruit slices were artificially inoculated with a cocktail of L. Treatment was more successful on melon followed by pear; however, no effect on apple products was noted. The study discussed above by Bai et al.

Typhimurium [ 96 ]. Raw materials used in the food industry which includes both crops and animals are at risk of microbial contamination. Pathogenic bacterial manifestations in agricultural animals can result in a reduction of quality of the food product or a reduction in yield due to the unsuitability of meat from infected animals. As an alternative to antibiotics, much research has been generated to investigate the suitability to treat pathogenic bacterial infections in agricultural animals with phage therapy [ 73 ].

Carvalho et al. Similarly, when a phage cocktail was administered to pigs inoculated with S. Typhimurium, there was reduction in the titre of S. IgE antibodies fixed to mast cells react with the complete virus or with viral components, triggering release of histamine and activation of slow reacting substance SRS-A and eosinophil chemotactic factor ECF-A. These act on blood vessels, smooth muscle and secreting glands to give the typical anaphylactic type reaction. Allergy to viruses usually results in a very localized anaphylactic reaction.

Furthermore this viral-mediated reaction is limited to a few virus species. Type II. The effects can be of two types:. This is the pathogenic mechanism in many viral diseases where anemia is one of the clinical manifestations. Antibody and complement bind to this infected cell and cause a lysis of that cell.

This is thought to be the major mechanism of viral-induced cell lysis. Type III. Although much rarer, some viral diseases may result in a generalized rather than localized tissue destruction.

This type of disease is a multi-system complement-dependent vasculitis in which immune complexes are deposited along the endothelial surfaces of blood vessels, stimulating inflammation and vascular wall damage.

The III reactions are known as Arthus-type reactions. The classical symptoms of this type of hypersensitivity are edema, polymorphonuclear leukocyte infiltration and hemorrhage. These are followed by secondary necrosis which reaches a maximum in hours. This type of hypersensitivity is due to precipitating antibody only, and requires a large amount of antibody.

The antibody is not fixed to the tissues. Histamine does not duplicate the reaction and antihistamines do not suppress the reaction. Type IV. This type of allergic reaction does NOT involve antibody. Sensitized T-lymphocytes react directly with viral antigen, usually that antigen expressed on the surface of an infected cell, producing inflammation through the action of lymphokines.

This leads to lysis of the infected cell. This is a delayed-type hypersensitivity which results in the Zinkernagel-Dougherty phenomenon. This is probably the second most common allergic reaction to viruses. During the course of virus replication, many viral components as well as by-products of viral replication accumulate in the cell. These are often cytotoxic e. The molecular mechanism of these toxins is not known in most cases. Only gross morphological defects can be observed generally. Some examples are:.

The construction of cDNA clones encoding large-size RNA molecules of biological interest, like coronavirus genomes, which are among the largest mature RNA molecules known to biology, has been hampered by the instability of those cDNAs in bacteria. Herein, we show that the application of two strategies, cloning of the cDNAs into a bacterial artificial chromosome and nuclear expression of RNAs that are typically produced within the cytoplasm, is useful for the engineering of large RNA molecules.

Finally, the complete genomic sequence of the infectious clone was determined and the cloned viral genome was shown to be identical to that of the parental virus. In summary, the generated infectious clone will greatly facilitate studies on individual genes and pathogenesis of CPXV.