Food-borne infectious diseases are a major global health concern. Enteric diseases are the second leading cause of child death worldwide killing nearly 1.7 million children every year.1
According to the World Health Organization (WHO), the bacteria: Campylobacter
and E. coli
O157:H7 are the three most prominent disease-causing food-borne contaminants.2
The development of a quick, low-cost, easy to use, portable food-testing device would be transformative in the establishment of adequate food safety programs throughout the developing world—diminishing the reliance on costly laboratory infrastructure.
Bacteria are routinely detected and identified by microscopy, colony-forming assay, PCR3
More recently bacteriophages have been used in a phage amplification assay5
and in fluorescence microscopy with labeled phages.6
These methods however are time-consuming, labor-intensive, and require specialized laboratory skills. There are rapid biosensor platforms being developed for microcantilever, surface plasmon resonance, quartz crystal microbalance and impedometric-based detection.7
However, these systems are dependent on the capture of the analyte on an interface.
Bacteriophages have several advantages over antibodies that are conventionally used as probes for bacterial detection. Bacteriophages are stable macromolecular assemblies that are relatively insensitive to temperature, pH, and ionic strength compared with antibodies. In fact, many phages can maintain their ability to infect for decades.8
They are also easy to produce by simple infection of their host bacteria whereas antibody production (monoclonal and polyclonal) is expensive and complicated.9
Bacteriophages initiate infection of their hosts by adsorption and then molecular recognition of the bacterial cell surface. The phage tails that bind to host cell surface polysaccharides or proteins mediate the recognition.10,11
Phage recognition of its host is commonly specific enough to differentiate between strains of the same species and this unique recognition makes bacteriophages an excellent choice as probes for selective detection of their host pathogen. Furthermore, bacteriophages are considered the most widely distributed biological entity in the biosphere, with an estimated population density of ~10 million/cm3
in any environmental niche where bacteria reside.12
We believe that this incredible biodiversity is a major strength of the intact phage approach.
Reporter bacteriophages are unique systems that have been developed for detection of bacteria exploiting the specific recognition of these viruses. A reporter bacteriophage carries a reporter gene that is delivered into the host bacteria upon infection and is expressed by the bacterial molecular machinery enabling their identification. Bacteriophages by themselves are incapable of expressing the gene and do not show signal until the gene is delivered into the host and thus a positive expression of the gene is a direct indicative of the presence of the host bacterium. Several reporter phages such as luciferase reporter phages (lux13
), ice nucleation reporter phages,15
fluorescent dye labeled phages,16
etc have been used for target organisms including Salmonella
Hagens et al. give a detailed account of use of reporter phages for the detection of food born pathogens19
while Smartt et al. describe the general application of this technology in a recent review.20
However, use of biosensors for bacterial detection has gained tremendous popularity for improved detection limits and possibility of developing point of care devices for fast and accurate assessment. Improving the strategy for bacteriophage immobilization on a biosensor platform has therefore become a field of active research in the recent years.
All previous literature discussing surface-immobilized bacteriophages for the capture of bacteria use partially-purified phage suspensions.21-24
Propagated phages and the resultant lysate are full of contaminants derived from the bacterial host, such as lipopolysaccharides (endotoxin), peptidoglycan fragments, flagella and proteins. These previous studies do describe some preliminary purification steps from the crude lysate. Bacteriophage purification and concentration by CsCl gradient, PEG precipitation, ultrafiltration, and ultracentrifugation are the most common methods. However, these methods are either not efficient enough to remove most contaminants from the preparation after one purification, are time-consuming or produce a low yield. Recent advances in bacteriophage purification are chromatographic methods. Ion exchange chromatofocusing is possible, but would requires determination of the phage pI and stability of the phage through a pH range for each phage system under study.25
Sephacryl S-500 size exclusion chromatography (SEC) has also been demonstrated,26
but most phages would be larger than its exclusion limit and would elute in the void volume with other large contaminants.
SEC by Sephacryl S-1000 proves to be an excellent, simple, and versatile method for purification of entities such as bacteriophages < 400 nm in diameter27
—which constitute a very large set of the known range of phage diversity. Concentrated phage preparations can easily be loaded onto the column and purified phage eluent collected automatically as is typically done by most FPLC systems. The separation is non-destructive and can occur under mild conditions (pH 7, room temperature, PBS eluent).
Previous work with unpurified T4 suspensions has shown that the use of covalent bonding in surface attachment gives a density of 18 ± 0.15 phages/µm2
We report here a substantial increase of phage surface density when chromatographically purified suspensions are rather used, resulting in an improved surface coverage for the purpose for capturing the host pathogen. This has in turn resulted in a marked improvement of E. coli
capture density. Phage surface clustering ultimately limits the T4 phage-immobilized surface’s ability to specifically capture its host bacteria. Nevertheless, this is to our knowledge the largest surface capture density of E. coli
reported using intact T4 bacteriophages. We extended this study to two other phage suspensions (P22 and NCTC 12673), which also show significant improvement in phage surface density.
Most importantly, such improvement of phage binding allowed a rigorous study of the surface attachment isotherm. Our analysis reveals that phage attachment to the surface does not obey the idealized Langmuir isotherm, but rather fits closest to the Brouers-Sotolongo isotherm,28
suggesting that a highly heterogeneous surface exists. We assert that phages initially attaching to the surface could be providing lower-energy sites for additional phage attachment, thus explaining the extensive surface aggregation, or clustering of phages, observed at higher phage titers. Finally, we have also applied these improvements to demonstrate the real-time capture of E. coli
using surface plasmon resonance (SPR) with a T4-immobilized surface.