The host specificity of phages makes them ideal candidates for use as recognition elements in bacterial diagnostic assays. Several phage-based methods have been developed to effect rapid detection of bacteria. These methods include the creation of genetically modified reporter phages and detection methods in which unmodified phages infect their host and amplify themselves to a detectable level which is measured by an end point assay such as the plaque assay. Alternatively, phages can be used to specifically lyse their host bacteria, releasing intracellular components which can then be detected.
Another phage-based approach to bacterial detection is to directly label the infecting phage itself. The resulting phages can then specifically tag bacterial cells, in a manner similar to antibodies, and may then be detected by several methods including epifluorescence microscopy, flow cytometry, confocal microscopy and fluorescent plate readers. Hennes and coworkers were the first to utilize labeled phages to detect bacteria. In their work, fluorescently stained phages were used as probes to label, identify and enumerate specific strains of marine bacteria and cyanobacteria. The results of the study indicated that the fluorescently labeled phages could be effectively used to detect and quantify specific groups of bacteria within mixed microbial communities.19
Following up on this work, Goodridge and coworkers created fluorescently labeled phages that could detect E. coli
In this approach, the double stranded DNA (dsDNA) contained within phage LG1 was labeled with the fluorescent nucleic acid stain YOYO-1™. Immunomagnetic separation was used to initially concentrate the E. coli
O157:H7 cells, which were then immersed in a suspension of the labeled phage. The suspension was subsequently filtered onto a black membrane and viewed under an epifluorescence confocal microscope, which showed that the target E. coli
O157:H7 cells had a “halo” like appearance, due to the bound fluorescent phages. When used in combination with flow cytometry, the assay was capable of detecting 104
CFU/ml in pure culture. In spiked food, the assay had a detection limit of 2.2 CFU/g in artificially contaminated ground beef following 6 hours enrichment, while between 101
CFU/ml of artificially contaminated raw milk were detectable after a 10 hour enrichment step. Other researchers used similar methods to effect detection of other bacterial species. For example, Mosier-Boss and coworkers labeled the dsDNA of phage P22 and used it to detect Salmonella enterica
serovar Typhimurium. Following phage infection, the researchers were able to visualize the labeled DNA inside of infected cells with the use of a fluorescent microscopic imaging system.20
Similarly, several researchers combined the reporter phage concept and labeling of phages to create genetically modified labeled phages, which displayed the green fluorescent reporter protein on the capsid surface.21,22
This technique allowed the detection of viable but non-culturable bacteria (VBNC) within one hour, if sufficient levels of bacteria were present.21,22
Phages have also been labeled with quantum dots and used to detect bacteria.23
One disadvantage of the above studies is the fact that they all required expensive instrumentation to detect the fluorescently bound phages. The use of such equipment, while allowing for extremely sensitive detection of bacterial cells, limits the use of such methods to a laboratory setting.
In this study, we attempted to address some of the limitations of previous labeled phage assays by creating several labeled phages by decoration of the external structural proteins of the phage with HRP, using a simple chemical process. The methodology was rapid and simple, and it is theoretically possible to label any phage using this technique. When compared to fluorescent labels, the use of an enzyme (HRP) to label the phages increased the sensitivity of the assay, especially when a luminescent substrate was used. Luminescent substrates have the widest dynamic range of all three (colorimetric, fluorescent and luminescent) classes of substrates.24
The Phazyme assay also required minimal equipment to read the test results. The Snap Valve™ devices that house the reagents for each Phazyme assay are designed to fit into a number of hand-held luminometers, and these devices are already widely used in the food industry for hygiene monitoring.
STEC infection may result from the ingestion of contaminated food or water or may be associated with animal contact.25
Reduction of food, water and environmental contamination by STEC is an important part of infection control, and detection of these pathogens in food and water samples represents the first step of an integrated control plan. In this study, Phazyme assays were created to detect E. coli
isolates from STEC serogroups O26, O103, O111, O145 and O157. The rationale for focusing on these serogroups is due to the fact that STEC is associated with a wide spectrum of illness ranging from uncomplicated watery diarrhea to hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS), which may result in death and many studies have confirmed that serogroups O26, O103, O111, O145 and O157 are highly associated with HC and HUS.26–31
In pure culture, the direct detection limits of the Phazyme assays ranged from 6.82 × 104 CFU/ml for serogroup O145 to 1.32 × 106 CFU/ml for serogroup O111 (). The direct detection limits of 4 of the 5 serogroups (O26, O103, O145 and O157) were within 0.7 log units, ranging from 6.82 × 104 CFU/ml for O145 to 3.85 × 105 CFU/ml for O103. The serogroup O111 Phazyme assay was the least sensitive of all of the assays, with a direct detection limit of 1.32 × 106 CFU/ml. One of the reasons for the differences in sensitivity between the O111 Phazyme assay and the rest of the assays may be due to the fact that the phage that was used in the O111 assay, phage 56, adsorbed with the lowest efficiency to O111 cells, when compared to bacterial cells from other serogroups (). For example, after 20 minutes, 69% of phage 56 was adsorbed to the O111 host, which corresponds to almost 9% less binding efficiency than the next nearest phage (ARI binding to O103 cells; 77.9%). Phage CBA120 adsorbed with the best efficiency to its host O157 cells with an efficiency of 93.1%. The binding efficiency of each phage to its host cells is directly relevant to the sensitivity of the assay, since the higher amount of target cell-phage will increase the amount of HRP that is present for detection. The isolation of phages that adsorb with higher efficiency to STEC serogroup O111 cells should increase the direct sensitivity of the assay.
When an 8 hour enrichment step was included, the sensitivity of 4 of the 5 Phazyme assays (serogroups O26, O103, O111 and O157) was 100 CFU/ml (). The one exception was the assay for serogroup O145, which was approximately 4 orders of magnitude less sensitive, with a detection limit of 104 CFU/ml. The reason for this is unknown. The lack in sensitivity was not due to inefficient adsorption of phage to the O145 bacterial surface, since after 20 minutes, 81% of phage AR1 adsorbed to the O145 cells, which is similar to the adsorption rates of AR1 for the O26 and O103 cells, 82.6% and 77.9% respectively (). The lack of sensitivity may have been due to the fact that the O145 IMS beads did not seem to be as effective at concentrating the bacterial cells as the other serogroup-specific IMS beads. For example, when IMS was conducted on E. coli cells belonging to STEC serogroups O26, O103, O111 and O157, a visible agglutination of the beads (due to multiple bacterial cells binding to more than one bead in solution) was observed. However, when IMS was conducted on O145 cells, the visible agglutination was not observed (data not shown). If the O145 IMS beads are inefficient at concentrating the bacterial cells, this would result in less bacteria present that could be labeled with phage, which would explain the lower sensitivity of the O145 Phazyme assay.
Interestingly, the O157 Phazyme assay displayed a distinct hook effect (). For any binding assay to give accurate results there must be an excess of the recognition element (RE) (antibodies, phages, aptamers, etc.,) relative to the analyte being detected. It is only under the conditions of RE excess that the dose response curve is positively sloped and the assay provides accurate quantitation. As the concentration of analyte begins to exceed the amount of the RE, the dose response curve will flatten (plateau) and with further increases may paradoxically become negatively sloped in a phenomenon termed the high concentration hook effect.32
Because the possibility exists that some samples may have analyte concentrations in excess of the RE, it is necessary to validate all samples by dilutional linearity analysis to establish if they are on the valid, positively sloped region of the curve or on the negatively sloped hook region of the curve. In this work, the fact that the labeling of the phages resulted in greater than a 1 log decrease in the concentration of infective phages, indicates that there is a high probability that the high concentration hook effect phenomena occurred at high bacterial concentrations. However, the hook effect only becomes an issue if the assay will be used to quantify the concentration of the target analyte. The Phazyme assays are designed to only detect and not quantitate STEC; as such, the hook effect should not constitute a problem as even the highest concentration of cells (103 CFU/ml) produced a signal that was much higher than the background ().
The specificity of the O26, O103 and O157 Phazyme assays were observed to be greater than 93% (). The O111 and O145 assays were less specific with 82.4% and 75.9% of the bacteria tested correctly identified. The lower specificity observed for these assays is directly related to the host range of the bacteriophages used in these tests. For example, both phage AR1 and phage 56 attached to several non-STEC isolates (), and this contributed to the lower specificity. In the future, phages that have less broad host ranges will be isolated and used in Phazyme assays for STEC serogroups O111 and O145.
The results of the food trials showed that the O157 assay was capable of very sensitive detection of STEC O157 cells in a variety of food and water samples. The results of the inoculated beef studies demonstrated the effects that food matrices can have on detection sensitivity. For example, the background of the rib steak samples was much higher than the background of the top round steak samples, which was probably due to the high fat content on the rib steak. Although this did not affect the sensitivity of the test (the Phazyme assay detected 100 CFU/100 cm2 in both samples), it is clear that the sample matrix must be taken into account when conducting the Phazyme assay.