Maite Muniesa and Juan Jofre
Bacterial virulence factors such as toxins are often encoded by bacteriophages. Among other examples, factors encoded by phages have been described in some of the emerging or re-emerging pathogens, including the pyrogenic exotoxin A production in group A streptococci (1), the cholera toxin in Vibrio cholerae (2), or enterotoxin production in enterohemorrhagic (EHEC) strains of E. coli (3).
Most described virulence factors in Shiga toxin (Stx)-producing E. coli strains are located in mobile genetic elements such as plasmids and bacteriophages. Stx, which are one of the most important virulence elements in Shiga toxin-producing E. coli (STEC), are encoded in the genome of temperate bacteriophages infecting E. coli and other Enterobacteriaceae (3-5).
Studies on Stx phages indicate that they are transmitted between different bacteria in vivo (6) and in vitro (7). Phages could also be transmitted extraintestinally, hence the observed presence of infectious Shiga toxin phages in sewage and in fecally contaminated rivers. Stx phages also show a higher persistence under natural inactivation and disinfectant treatments in aquatic environments (8-10).
This background shows that phages or lysogenic strains carrying Stx2 phages might be the natural reservoir of Stx2 genes and that lysogenization could be the main cause of the emergence of STEC strains, as suggested by several authors (3,7,11). It has also been suggested that lysogenization/conversion processes could take place in food and water and probably inside the human and animal gut. Ingestion of Stx2 phages could produce conversion of non-Stx2-E. coli strains, present inside the gut and producing new pathogenic strains. To control these phenomena, it is first necessary to gain more information about the distribution of Stx phages in the environment.
For this purpose, a method of detecting Stx2 phages present in environmental water samples has been developed. The particularity of this method is that it allows detection of all (infectious and noninfectious) Stx2 phages in a water sample; in a second stage, the method allows detection of those phages able to infect and replicate on E. coli O157:H7. Although this method has been applied to Stx2 phages able to infect E. coli O157:H7 (8), it is also applicable to detection in the natural environment of other genes carried by other bacteriophages and other bacteria.
For direct isolation of phages carrying the gene from a water sample, phages are partially purified by ultracentrifugation of different volumes of the sample; the phage DNA is extracted and amplified by polymerase chain reaction (PCR)-nested PCR and then confirmed by hybridization and/or sequencing. For detection of infectious bacte-riophages carrying the gene, a water sample (e.g. sewage or river water sample) is used to infect a culture of E. coli O157:H7 (ATCC 43888, which does not contain the Stx2 gene). Phages present in the water sample infect the strain and multiply and can be recovered from the supernatant of the enrichment culture. After recovery of phages, phage DNA is extracted, and the Stx2 gene is amplified by PCR and nested PCR.
This approach allows determination of the presence of converting phages in water as well as their quantification. To enumerate Stx2 phages from a water sample, several enrichment cultures are performed, each one infected with a different volume of treated water samples. Results of positive or negative nested PCR amplification for each volume of sample allow enumeration of Stx2 phages by applying the most probable number (MPN) technique. Although the number obtained is a statistic, this approach yields information about the amount of Stx2 phages in a given sample.
The method allows analysis of the supernatant of the enrichment culture to evaluate the process of transduction of the genetic character understudy (Stx or other proteins) carried by phages present in the sample. Detecting the presence of the protein codified by the gene being studied in the supernatant of a culture after infection with phages present in the environment is another extension of the protocol described here, which could help to evaluate the rate of phage conversion in natural environments.
2.1. Obtaining Phages Directly from Sewage
2. 2X Phosphate-buffered saline (PBS): 8 g NaCl, 0.2 g KCl, 0.2 g KH2PO4, 1.15 g Na2HPO4.
2.2. Culture of the Host Strain and Phage Infection
1. A host strain negative for toxin production (e.g., E. coli O157:H7, ATCC 43888).
3. Luria broth (LB) basal medium: 10 g peptone, 5 g yeast extract, 10 g NaCl in 1 L distilled water; or Luria agar (LA) made by addition of 7 or 15 g agar-agar. Adjust the pH so that after sterilization it will be 7.2 ± 0.2. Sterilize in the autoclave at 121 ± 1°C for 15 min.
4. 0.1 M Calcium chloride solution: dissolve CaCl2 in the water while heating gently. Cool to room temperature (RT) and filter-sterilize through a 0.22-^m pore size membrane filter. Store in the dark at 5 ± 3°C for not longer than 6 mo.
5. Samples: although the protocol is developed for environmental water samples, other kinds of samples could be used.
6. Filters of 0.22-^m pore size of polyvinylidene difluoride (PVDF; a low-protein-binding membrane; Millex-GV, Millipore).
7. DNase I (final concentration 10 U/mL) and RNase (final concentration 30 U/mL).
2.3. DNA Extraction
3. 0.5% Sodium dodecyl sulfate (SDS).
4. Phenol/chloroform/isoamyl alcohol (25:24:1, v/v).
6. 3 M Sodium acetate.
2.4. Polymerase Chain Reaction
1. PCR amplification kit.
2. Custom-made oligonucleotides:
a. First-round PCR
Upper primer, 5'-GCGTTTTGACCATCTTCGT-3'. Lower primer, 5'-ACAGGAGCAGTTTCAGACAG-3'.
b. Nested PCR
Inner upper primer, 5'-TAATACGGCAACAAATACT-3'. Inner lower primer, 5'-TGATGAAACCAGTGAGTGA-3'.
5. 2 mg/L Ethidium bromide.
6. Gel loading buffer: 40% sucrose, 0.25% bromophenol blue.
7. Standard molecular weight markers.
2.5. Labeling of the Probe With DIG
1. 1 mM Digoxigenin (DIG)-11-dUTP.
2. Nucleotide mix for labelling: 10 mMdATP, 10 mMdGTP, 10 mM mMdCTP, 7 mMdTTP.
3. PCR amplification kit and primers.
4. PCR Purification kit (e.g., QIAquick™, Qiagen, Hilden, Germany).
1. Nylon-N+ membranes.
2. Custom-made probe: 5'-ATGACAACGGACAGCAGTTATACCAC-3'. The PCR amplimer obtained either with first-step PCR or nested PCR could also be used as a probe.
3. Buffer 1: 100 mM maleic acid, 150 mM NaCl, pH 7.5, with solid NaOH. Sterilize by autoclave. Store at RT.
4. 20X SSC: 3 M NaCl, 0.3 M sodium citrate, pH 7.0. Sterilize by autoclave. Store at RT.
5. Hybridization buffer: 6X SSC, 50 mM NaPO4, 0.5% SDS.
6. Washing buffer: buffer 1 + 0.3% Tween-20. Keep at RT.
7. Blocking reagent stock solution: 10% (w/v) of blocking reagent (Boehringer Mannheim) in buffer 1 with several 30-s heat pulses in the microwave (3 or 4 min total). Sterilize by autoclave. Store at RT or at 4°C.
8. Buffer 2: blocking reagent stock solution diluted 1:10 in buffer 1. Store at -20°C for no more than 2 mo.
9. AntiDIG antibody.
10. Buffer 3: 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 50 mM MgCl2.
11. NBT/BCIP (4-nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate; Boehringer Mannheim).
12. Plastic bags and hybridization tubes.
13. Hybridization oven or thermal water bath.
14. Thermal sealer.
2.7. Detection of the Protein Encoded by the Gene Carried by Bacteriophages in the Supernatant of the Enrichment Cultures
1. 10-K Cutoff filtration membrane microconcentrators (Microsep, Millipore).
2. Material for the preparation of the polyacrylamide gel (Table 1).
3. Electrophoresis supply and vertical electrophoresis apparatus.
4. SDS gel loading buffer: 50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.05% bro-mophenol blue, 0.1% 2-|3-mercaptoethanol. Store at RT.
5. Running buffer: 25 mM Tris-HCl, 250 mM glycine, 0.1% SDS. Dissolve and store at 4°C.
6. Low-range stained SDS-PAGE standard, commercially available.
7. Semidry transfer cell (Trans-blot Semi Dry, Bio-Rad).
8. Nitrocellulose membranes (PROTRAN, Schleicher & Schuell).
9. Transfer buffer: 24 mM tris base, 192 mM glycine, and 20% methanol.
10. Blot absorbent filter paper (thick).
11. Blocking reagent: 2 mM Tris-HCl, 28 mM NaCl containing 0.02% Tween-20 and 3% serum albumin.
12. Antibody against Stx2-A subunit. Antibody is obtained from hybridoma cell line 11E10 (ATCC CRL1907).
13. Anti-mouse alkaline phosphatase-conjugated antibody.
14. Washing buffer: 2 mM Tris-HCl, 28 mM NaCl containing 0.02% Tween-20.
15. 5X Base buffer number 3: 1 M Tris-HCl, pH 9.6, 5 M NaCl.
16. Detection buffer for immunoblot: 20% base buffer #3, 2% 2 M MgCl2. Dissolve and use immediately.
17. NBT/BCIP (Boehringer Mannheim).
The method described here can be applied to determine the presence of the gene either in DNA extracted from phages directly isolated from sewage (see Subheading 3.1.) or in phages that have infected a bacterial strain and are isolated from the supernatant of the enrichment cultures after infection (see Subheading 3.2.).
3.1. Obtaining Phages Directly From Sewage
1. Bacteriophages were recovered from sewage and partially purified as follows.
2. First, 100, 10, and 1 mL of sewage were centrifuged for 3 h at 48,000g.
3. The pellet was resuspended in 0.25 N glycine buffer, pH 9.5, shaken at 4°C for 30 min to disrupt the bacteriophage clumps, and then buffered to pH 7.4 with PBS 2X and again centrifuged at 12,000g for 20 min.
4. The supernatant was further centrifuged for 1 h at 171,000g.
5. The pellet was finally dissolved in 100 ^L of PBS.
Material for Preparing the Polyacrylamide Gel
Material for Preparing the Polyacrylamide Gel
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