Rapid and sensitive detection of human and animal pathogenic bacteria is of great importance to many fields of medicine, food manufacturing and production of pharmaceuticals. Traditional detection methods depend on selective culturing the organism of interest, followed by biochemical characterization. The procedure is labor-intensive and, more importantly, very time consuming. Molecular methods such as PCR have provided rapid diagnostics, however, such procedures demand skilled labor in execution, and even if automated, the necessary machinery may be expensive. Another important disadvantage is that these screenings do not provide information about the viability of the target cells.
Bacteriophages are bacterial viruses with a very high host-specificity, specifically infecting single species or members of a genus. This intriguing biological specificity has led to the development of several phage-based detection and identification methods and strategies.1–3
Reporter bacteriophages are genetically modified to transduce a reporter gene into the bacterial target cells. They are of particular interest, as they offer a highly specific, low-cost and easy to use means of detecting viable and metabolizing target bacteria. Of these, luciferase reporter phages (LRPs) are the best known.4
They encode bacterial or firefly luciferase genes which are expressed in infected target cells and lead to the formation of a luminescence signal which can de detected. LRPs have been described for several enterobacteria, Listeria, Bacillus and Mycobacterium.5–17
Another type of reporter gene used is the ice nucleation gene (ina
) which was introduced into Salmonella phage FelixO1.8,18
These two gene products have in common that relevant food samples are unlikely to contain background activity that could obscure test results. Development of useful LRPs depends on the availability of a phage with a broad host range suitable to detect the majority of targeted strains. Listeria bacteriophage A511 is a member of the Myoviridae
, and has a large, terminally redundant and non-permutated genome of 134.5 kb.19
This phage features a very broad host range within the genus Listeria and was used to construct A511::luxAB
for rapid and sensitive bioluminescence-based detection of viable Listeria cells from food samples.10,11
However, the Vibrio harveyi
LuxAB fusion protein expressed by phage infected cells is unstable at temperatures above 35°C, which may limit its use in other phage-host systems. Moreover, emission of bioluminescence from phage-infected cells is only transient, i.e., must be measured in a very short time window after addition of the substrate. This is not due to enzyme stability or substrate limitation (substrate can be added at will), but by the amount of FMNH2
co-factor present in the reaction mixture, i.e., the infected cells. Depletion of this co-factor is rapid, resulting in a flash of light which lasts only seconds after substrate addition to the sample.10
This complicates application protocols and limits the detection threshold, as longer incubation will not yield a stronger signal. For measurement of bioluminescence, single-tube luminometers with manual or semi-automated substrate addition can be used, which is time consuming and results may be difficult to reproduce. Therefore, handling of larger sample numbers requires plate readers equipped with automated injectors and sensitive photo-multiplier optics.
To remove some of these limitations, our aim was to develop an alternative reporter phage approach designed for maximum ease-of-use, which would permit simple end-point titration of enzyme turnover, and a standard photometric 96-well plate reader. The CelB enzyme from the hyperthermophilic archaeon Pyrococcus furiosus
is an extremely thermostable glycosidase,20
which features highest activity at a pH of 5–5.5, and a temperature of 102–106°C.20,21
It exhibits both β-glycosidase and β-galactosidase activities, thus enabling possible use of a wide range of substrates for detection of its activity in a given sample. By incorporating celB
into Listeria phage A511, background activity of any other enzymes in a given sample can be eliminated by heating after production of the enzyme by the phage-infected target cells.