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Reporter bacteriophages for detection of pathogenic bacteria offer fast and sensitive screening for live bacterial targets. We present a novel strategy employing a gene encoding a hyperthermophilic enzyme, permitting the use of various substrates and assay formats. The celB gene from the hyperthermophilic archaeon Pyrococcus furiosus specifying an extremely thermostable β-glycosidase was inserted into the genome of the broad host range, virulent Listeria phage A511 by homologous recombination. It is expressed at the end of the infectious cycle, under control of the strong major capsid gene promoter Pcps. Infection of Listeria with A511::celB results in strong gene expression and synthesis of a fully functional β-glycosidase. The reporter phage was tested for detection of viable Listeria cells with different chromogenic, fluorescent or chemiluminescent substrates. The best signal-to-noise ratio and sufficiently high sensitivity was obtained using the inexpensive substrate 4-Methylumbelliferyl-α-D-Glucopyranoside (MUG). The reporter phage assay is simple to perform and can be completed in about 6 h. Phage infection, as well as the subsequent temperature shift, enzymatic substrate conversion and signal recordings are independent from each other and may be performed separately. The detection limit for viable Listeria monocytogenes in an assay format adapted to 96-well microplates was 7.2 × 102 cells per well, corresponding to 6 × 103 cfu per ml in suspension. Application of the A511::celB protocol to Listeria in spiked chocolate milk and salmon demonstrate the usefulness of the reporter phage for rapid detection of low numbers of the bacteria (10 cfu/g or less) in contaminated foods.
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.
The construction of plasmid pBS511-F3s-celB is schematically represented in Figure 1. Since plasmid pBS511-F3s has an insert in the multiple cloning site, successful insertion of celB was detected by formation of blue colonies on agar plates supplemented with X-gal after transformation of E. coli XL1B. This also confirmed functionality of the CelB protein. Correct sequence insertion in plasmid pCK511F3s-celB isolated from L. ivanovii WSLC 3009 was determined by restriction fragment analysis, since no CelB activity could be detected at the lower growth temperature of 30°C.
We employed homologous recombination between a replicating plasmid and the broad host range, virulent Listeria bacteriophage A511 DNA during infection of Listeria host cells to insert the coding sequence for the hyperthermophilic CelB glycosidase amplified from genomic DNA from the archeon Pyrococcus furiosus into the phage genome. The procedure worked exactly as predicted from previous experience.10 Soft-agar overlay plates of L. ivanovii infected with 300–1,000 pfu/plate were prepared. These were first incubated at room temperature to allow formation of large plaques, incubated at 50°C for 30–60 min (allowing the enzyme to be active), followed by incubation at 37°C for 5 h, and storage at 4°C overnight to allow color formation. This procedure enabled us to identify very few light blue, presumably CelB-positive plaques. Their frequency correlated well with the frequency of homologous recombination (5 × 10−4) previously reported for construction of A511::luxAB.10
Phage DNA of a single well isolated blue plaque was used for PCR amplification of the celB containing region, and sequencing of the PCR product confirmed correct insertion and sequence of the recombinant phage.
Detection of 1.2 × 103 viable Listeria ivanovii WSLC 3009 cells (125 µl of a 104 cfu/ml suspension) by phage-transduced CelB β-glycosidase activity was generally possible at all tested pH values, temperatures, and with all substrates and concentrations used. However, best results were obtained at a pH of 5.0, yielding highest signal-to-noise ratios, regardless of the other parameters (Fig. 2A). This finding corresponds well to the pH optimum of the native enzyme.20,21 Figure 2 shows the results obtained at pH 5.0 and 75°C incubation temperature, which showed a slower increase of the signal but higher signal-to-noise ratios after prolonged incubation. This was typical for the readings obtained at the lower incubation temperature, although the optimum temperature for CelB activity is about 100°C. Figure 2B also demonstrates that while the relative signal intensity increased fastest at 95°C, the disproportionally increasing background noise resulted in an overall weaker signal ratio, compared with samples incubated at lower temperatures for longer time periods.
A similar phenomenon was observed with respect to substrate concentration. Background noise increased disproportionally to the signal at the highest substrate concentration of 0.45 mM, less so at 0.3 mM, and the least interference was observed at 0.15 mM. Again, the most favorable signal-to-nose ratio was observed at extended incubation of more than 6 h. Since a rapid detection analytical protocol should be completed in a reasonably short time, a cut-off of 6 h was chosen to permit Listeria detection within one working day after prior enrichment in selective medium, or within 24 h of receiving the sample. Thus, standard assay conditions were set as incubation at 85°C, pH 5.0 and 0.3 mM MUG as substrate to detect CelB activity. All subsequent experiments were performed using these optimized parameters.
Different concentrations of target cells were assayed using the optimized conditions. A signal-to-noise ratio of at least 2 or higher (>2) after 6 h of enzyme activity was considered as significant and indicative of viable bacterial target cells in the sample. Figure 3 shows the results obtained with L. monocytogenes Scott A from 4 × 103 to 2 × 104 cfu/ml; signal ratios above the threshold >2 were only obtained when the concentration of the cell culture used was at least 6 × 103 cfu/ml.
Detection of viable Listeria cells after A511::celB infection was also possible with the chemiluminescent Galacto-Light Plus™ system. Figure 4A shows the increase in signal intensity compared with the background noise over the first 60 min while this ratio dropped after this point in time. This reduction was even more pronounced at the higher incubation temperature of 95°C. Changing the second parameter (pH) did not influence this effect (data not shown). Figure 4B shows the number of cells that can be detected with this assay at the optimal signal-to-noise ratio time-point of 1 h. While large numbers of cells generate a very strong signal compared with other tested substrates the small amount of enzyme active in more diluted cell-samples does yielded signal-to-noise ratios of approximately 3 in samples containing 70 µl of a 5 µ 104 cfu/ml suspension, and a ratio of ~2 in the case of samples containing 70 µl of a 5 × 103 cfu/ml suspension.
Listeria cells expressing transduced celB could also be detected with several other chromogenic β-galactosidase and β-glycosidase substrates, However, detailed data are not shown here because these substrates did not perform well and yielded poor signals, likely because the high incubation temperatures are unsuitable for these substrates. We conclude that MUG and Galacto-Light based assays are the best currently available CelB assays in terms of substrate stability, sensitivity and signal strength.
These experiments were performed using the MUG assay as it was slightly more sensitive in detecting low numbers of target cells and is easier to perform and less expensive than the chemiluminescence assay. It must also be noted that selective enrichment of Listeria spiked food samples should be performed using non-chromogenic Listeria enrichment broth rather than the more commonly used Fraser media. The aesculin glycoside contained in the latter shows very strong fluorescence when excited with UV light, which completely masked the signal obtained in the CelB-MUG assay, even after dilution of the primary enrichment with fresh non-selective media. Moreover, the dark brown color that is formed by a reaction of the aesculin breakdown product aesculetin (6,7-dihydroxycoumarin) with the iron salts present in the Fraser medium also interfered with optical readings.
The salmon and chocolate milk samples were analyzed for Listeria contamination using the A511::celB MUG-assay after 24 and 48 h selective enrichment, followed by a 2.5 h or 5.5 h second enrichment in ½ strength BHI, and 3–6 h phage infection. All samples spiked with 10 cfu/g tested positive after 48 h enrichment, and contamination levels of 1 cfu/g were detected in chocolate milk at all variations of the assay. In contrast, the same contamination rate did not produce a positive signal in the salmon samples (Table 2). Interestingly, increasing the amplification incubation in ½ strength BHI from 2.5 to 5.5 h after 24 h selective preenrichment significantly increased the signals from chocolate milk samples, whereas the signal from salmon contaminated with 10 cfu/g Listeria cells was positive only after 2.5 h amplification. Because of the signal strength increase in samples incubated for 5.5 h after 24 h enrichment only 5.5 h incubation was used for samples enriched for 48 h.
We report the construction of reporter bacteriophage A511::celB, which introduces the gene for an extremely thermostable glycosidase from Pyrococcus furiosus into phage-infected Listeria cells. Expression of celB from the phage late genes operon is placed under control of the strong cps gene promoter,10 and synthesis of the enzyme in the target cells was followed by detection of enzyme activity in these phage lysates. The assay allows rapid and sensitive detection of viable Listeria spp both in vitro and in artificially contaminated food samples. Interestingly, CelB features a relaxed substrate specificity; it can hydrolyze both β-glycoside and β-galactoside bonds, therefore permitting the use of a range of different commercially available colorimetric, fluorescent and chemiluminescent substrates for detection. The extreme thermostability of CelB permits convenient elimination of any endogenous enzymatic activity, and the long half-life of the enzyme at the lower temperatures of the reporter assay resulted in long lasting activity and signal strength increase over prolonged incubation periods.
Despite these favorable properties, the relatively low ratio of signal-to-noise ratio and the increase of the latter with extended incubation prevented direct detection of less than 5 × 103 cfu/ml, which in the assay format we used was 420 cells per well. In artificially contaminated foods, A511::celB allowed detection of 10 cfu/g L. monocytogenes after 24 h of enrichment within less than one day, whereas very low counts (1 cfu/g) could only be detected in chocolate milk. The finding that the food matrix has a significant influence on the detection of Listeria from such samples corresponds well to reports on bioluminescence-based reporter phage, and also to surface plating on selective media.11 The most likely explanation is that the different food/enrichment broth mixture offer different growth conditions to Listeria, not only with respect to the food itself but also with respect to its endogenous microflora.
We found that the sensitivity of the A511::celB assay, here defined as the lower limit for direct detection of Listeria cells from pure culture, using the MUG assay was quite similar to that of luciferase reporter phage A511::luxAB.10 As few as 750 cells could be detected in a single well. This result was surprising, since continuous CelB hydrolyzing activity was expected to enhance the read-out of the fluorescent dye assay, proportional to a longer reaction time. However, as mentioned above, this effect was apparently largely compensated by the simultaneously increasing background signal (the “noise”) in the MUG assay. However, with respect to the available substrates for CelB activity monitoring, MUG was still found to be superior to the other color-forming and also chemiluminescent compounds. Although as few as 420 cells per well could be detected using the chemiluminescence substrate, its apparent heat instability made it less suitable for use with an enzyme requiring high incubation temperature. A specifically designed substrate combining high sensitivity with greater thermostability would allow optimization of detection sensitivity. Assay times may possibly be shortened for practical reasons. The detection limit in terms of cells needed to generate adequate signal levels is quite satisfactory. In the food experiments, Listeria were taken from enrichment cultures but the plate format limits the amount of sample that can be added. If Listeria cells were to be separated from samples of interest, the total turnover time for detection could possibly be reduced significantly. One method to achieve this is immuno-magnetic separation (IMS) or even better CBD-based separation,22 which allows the rapid concentration of target cells from large sample numbers. These physical approaches may also be designed to provide added specificity, which would allow targeted detection of L. monocytogenes if desired.
The CelB-mediated signal after phage-infection of bacterial cells was significantly higher at 35°C incubation than at 30°C (data not shown). This difference (>5x) appears greater than increased protein synthesis at the elevated temperature would explain. A direct relationship between increased protein activity and elevated temperature has previously also been reported for Pyrococcus enzymes made in E. coli.23 The authors attribute the increased activity to better folding of the protein at the higher temperatures; which may also be the case for Listeria.
The use of celB as reporter gene features advantages such as its small size and the selection of commercially available substrates the enzyme can process. However, instability most of the substrates at the elevated incubation temperature and the associated proportional increase of the signal together with the background noise currently limit full exploitation of the unique properties of this unusual enzyme. This may be circumvented by the development of heat-stable β-glycoside substrates, or by the use of less thermophilic (but still thermostable) reporter enzymes featuring optimum activity in a 50–60°C range.
In summary, our study shows how versatile the use of such thermostable reporter genes in a phage can be, in terms of substrate and detection platforms. Phage infection, enzyme substrate conversion and signal recording are independent from each other and may be performed at different time points. With sequence data of numerous thermophilic organisms available, enzymes/phage systems can be optimized and such systems may prove to be superior not only in versatility and ease-of-use, but also in sensitivity.
All chemicals were from Sigma Aldrich unless otherwise specified. Restriction enzymes were purchased from Fermentas and Proofstart DNA polymerase was purchased from Qiagen (Hilden, Germany).
All organisms and plasmids used for construction of A511::celB and subsequent testing are listed in Table 1. E. coli was grown in LB medium at 37°C. Listeria was grown at 30°C in ½ strength BHI broth. Agar was supplemented at 1.5% and 0.4 percent for agar plates and soft agar respectively. Antibiotics, for selection of transformants, were added at 7.5 µg/ml in the case of chloramphenicol for both E. coli and Listeria, and ampicillin (160 µg/ml) for selection of pBluescript-derived vectors.
Plasmid pCK511-F3s-celB for homologous recombination and integration of the celB via double crossover upstream and downstream of the target sequence was constructed analogously as previously described for pCK511-F3s-luxAB.10 Instead of the end modified luxAB fusion gene, the celB sequence equipped with an optimized Listeria ribosome binding site and 50 nucleotides from the SnaBI site to the 3′end of the cps gene added at the end was inserted into SnaBI linearized and de-phosphorylated pBS511-F3s, providing the flanking regions required for homologous recombination with phage DNA.10 This large fragment was obtained in a two-step PCR, using plasmid pLUW511 20 as a template. It should be noted that the sequence of the celB-gene present in plasmid pLUW511 contains three additional bases immediately after the start codon introduced during initial construction of the plasmid. The PCR procedure as described here was designed to remove these bases and completely restore the wild type gene sequence.
The first PCR step (5 min denaturation at 95°C, followed by 25 cycles of 94°C, 35 sec denaturing, 60 sec annealing at 52°C, 100 sec extension at 72°C) was performed with primers O2 5′-CTG TAA AAA ACG TTC ATA GCA ACT AAG AGG AGG TAA ATA ATA TGA AGT TCC CAA AAA ACT TCA TGT TTG GAT ATT CTT GG (RBS and Start codon are bold, and the 5′ end of celB is underlined)- and P2 5′-TTA CTA CTT TCT TGT AAC AAA TTT GAG GTC TGC complementary to the 3′end of celB, including 2 consecutive stop codons. The second PCR (with identical conditions) was performed using primers Q2 5′-GTA TTA GAA ACG TTA AAT ATA TTC CTG TAA AAA ACG TTC ATA GCA ACT AAG, with the underlined portion completing the 3′ distal 50 nucleotides of A511 cps, and primer P2. Primers P2 and Q2 were 5′-phosphorylated to ease ligation into linearized pBS511-F3s. This plasmid was transformed into E. Coli XL1B. The celB-cps fragment for ligation into the exchange vector was recovered from a 0.8% agarose gel after electrophoresis of EcoRI-BamH1 digested plasmid using the QUIAEX II gel extraction kit (Qiagen).
Plasmid pCK511-F3s-celB was purified from E. coli and electroporated into Listeria ivanovii strain WSLC 3009. To recombine the modified celB into the 3′-locus of the A511 cps gene, L. ivanovii WSLC 3009 (pCK511-F3s-celB) was infected with wild-type A511, using the same procedure and conditions as described earlier in reference 10.
Sterile-filtered lysates containing recombinant phage were plated on wild-type WSLC 3009 at levels resulting in 100–1,000 plaques on standard soft-agar overlay plates supplemented with 80 µg/ml X-gal. After overnight-incubation at room temperature, plates were shifted to 50°C for 30–60 min to boost enzyme activity without inactivating the phage particles. Plates were incubated at 37°C for an additional 5 h and stored in a fridge overnight to let color develop. Well-isolated, single blue-colored plaques were then picked, phages eluted in 10 µl SM buffer, and re-plated on the same host. Finally, 10 phage isolates were tested for transducing CelB activity using a modified form of the Miller β-galactosidase assay.28 One ml of exponentially growing host cells (OD600 approximately 0.5) were infected with the phage isolates at an MOI (multiplicity of infection) of 0.1, and incubated at 35°C for 2 h to allow expression of phage genes. Cultures were then heated to 95°C for 15 min to inactivate any Listeria background enzyme activity, and subsequently held at 75°C. 0.2 ml of a 0.1 M solution of the colorimetric substrate ortho-nitrophenyl-β-galactoside (ONPG) was added, and the reaction stopped after 15 min by adding 50 µl 2 M Na2CO3 to the tubes.
Stocks of A511::celB were prepared as previously described in reference 10, further purified by CsCl density gradient centrifugation29 in order to remove any contaminating CelB activity, and stored at 4°C until further use.
The concentration of phage required for infection of all cells present in any given volume (and maximum reporter gene transduction into susceptible host cells) has been determined earlier.10 Therefore, 3 × 108 pfu/ml of A511::celB were added to samples, and incubated for 150 min at 35°C before heating, addition of substrate and signal measurements.
The parameters for optimum activity of the native CelB enzyme20,21 were used as guidelines to determine the best conditions for different enzyme/substrate combinations with respect to rapid detection of CelB-producing Listeria, aimed to obtain a low signal-to-noise ratio. To produce a stock test solution of phage-transduced enzyme in Listeria, log-phase cells of L. ivanovii WSLC 3009 were adjusted to 3 × 106 cfu/ml in 1 L of ½ BHI, and A511::celB was added at 3 × 108 pfu/ml. Phage infection and gene expression was allowed to proceed for 150 min at 35°C, followed by heating the mixture to 100°C for 10 min to inactivate all other, heat-labile enzymes. The mixture was then stored at −80°C in 1 ml aliquots for the subsequent experiments. The following single parameters were evaluated: pH 4.5–6 (adjusted by addition of HCl), temperatures (75, 85 and 95°C), and substrate concentrations (0.15, 0.3 and 0.45 mM of 4-Methylumbelliferyl-α-D-Glucopyranoside (MUG), followed by an iterative series of experiments aimed to optimize the individual values for maximum enzyme activity under the tested conditions. Phage-only and cells-only samples served as controls. For signal measurements, 125 µl aliquots of each sample were placed in white 96-well plates (Nunc, Denmark), and 75 µl Na2CO3 was added per well to stop the reactions. Fluorescence of the MUG metabolite was measured at 366 nm, using a semiautomated multilabel counter (Victor 3, Perkin-Elmer).
All experiments were performed in triplicate, and the assays parameters were also independently verified using another L. monocytogenes strain, ScottA serovar 4b (results not shown).
Detection of CelB glycosidase or galactosidase activity was also tested with the chromogenic (colorimetric) substrates ONP-Glu, ONP-Gal, X-gal and CPRG. All assays were performed at different incubation temperatures (75–85°C) and pH values (pH 5–6) in the Miller β-galactosidase assay.28 We also evaluated a highly sensitive chemiluminescent β-galactosidase assay (Galacto-Light Plus™, Applied Biosystems) for compatibility with the requirements of the CelB enzyme. The pH of the supplied buffer was adjusted to pH 5.5. At 150 min after phage infection, 70 µl the samples were mixed with 245 µl reaction buffer and incubated at different temperatures from 65–85°C for 45 min. Samples were then cooled to room temperature, and 90 µl aliquots were transferred to white 96 well plates. Luminescence was detected with a plate reader (Victor 3, Perkin-Elmer) following injection of Signal enhancing solution (Applied Biosystems) into the sample wells.
Smoked salmon and chocolate milk were purchased from local retailers and initially screened for contamination with Listeria according to EN ISO 11290 part 1:1997 30 or IDF standard 143A:1995.31 Then, samples of 25 g each were transferred into sterile polyethylene bags and spiked with L. monocytogenes Scott A at levels of 1 or 10 cfu/g by addition of 1 ml of appropriate dilution prepared from overnight culture of the organism. After 3 h incubation at 4°C, samples were homogenized in 225 ml Listeria selective enrichment broth using a stomacher laboratory blender, and incubated for 24 or 48 h at 30°C. Then, 1 ml samples were transferred to 4 ml 0.5x BHI broth and incubated at 37°C for 2.5 or 5.5 h. Phage A511::celB was added at 3 × 108 pfu/ml, followed by further incubation for 150 min at 35°C. For CelB activity measurements, sample pH was adjusted to 5.0, MUG was added (0.3 mM), and the samples were incubated at 85°C for 180 or 360 min before fluorescence was measured. All experiments were performed in triplicate.
We wish to thank the laboratory of Prof. Willem de Vos of Wageningen University for kindly providing plasmid pLUW511 with the celB sequence.
No potential conflicts of interest were disclosed.