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Verocytotoxigenic Escherichia coli (VTEC) is a serious pathogen of considerable public health concern worldwide. Infection is usually characterized by bloody diarrhea and can be life threatening due to the subsequent development of hemolytic-uremic syndrome mediated by verocytotoxins (VTs), of which there are two forms, VT1 and VT2. In almost all cases, the VT genes are carried on temperate bacteriophages (VT phages). Although E. coli O157 is the most commonly isolated VTEC serogroup in the United Kingdom, North America, and Japan, more than 30 disease-causing non-0157 VTECs have been described (1) and over 100 serotypes are capable of producing VT (6). VT production has been observed in other members of the Enterobacteriaceae, including Enterobacter cloacae (8) and Citrobacter freundii (12), but was first described in Shigella dysenteriae as Shiga toxin (3). The localization of vt genes on a bacteriophage was first described by Smith et al. (13), but their acquisition by pathogenic E. coli strains remained anomalous because only nonpathogenic (rough) E. coli strains could apparently be infected with VT phage. Previously, the vt₂ gene of a bacteriophage (24_B), isolated from an E. coli O157 strain, had been inactivated by insertion of a selectable marker (kanamycin resistance) (10). This provided an ideal opportunity to investigate the host range of a lysogenic VT bacteriophage and thus its potential to transfer the ability to produce VT between E. coli and related gram-negative bacteria.

The host range of this recombinant VT2 phage (24_B::Kan) was determined by infection of pathogenic and commensal strains of E. coli and other Enterobacteriaceae strains from human and animal sources. Lysogens were detected by spreading phage-infected cultures of the host bacteria (100 µl) onto Luria-Bertani Miller (LB) agar (Difco) plates containing kanamycin (50 µg ml¹).

As it is clear that some phage infections create lysogens and do not result in a lytic infection, plaque assays may not necessarily detect all infectious phage particles. Induction of the VT phage lytic cycle is RecA dependent (7). RecA plays a central role in the SOS response of E. coli, during which phage-mediated lysis is induced. The recA441 mutant E. coli K-12 strain, DM1187 (5), was used to detect all free phage particles. This mutation results in constitutive activation of RecA protease in the absence of induction. This leads to inactivation of the phage immunity repressor, preventing maintenance of lysogeny and forcing the phage into the lytic cycle. Phage stocks were prepared in this strain and stored at 4°C. Strain DM1187-rif was created by successive passage in increasing rifampin concentrations (5 to 500 µg ml¹). An indirect plaque assay was developed using this DM1187-rif strain, enabling the detection of all free infectious phage particles in any infection mix. Briefly, DM1187-rif was added, as an indicator, to a culture previously infected with phage. Rifampin (300 µg ml¹) was incorporated in the soft agar overlay to select for the indicator host. Lysogens from this infection mix, which could not be of DM1187 origin, could be detected on LB agar (Difco) containing kanamycin (50 µg ml¹). This allowed the detection of all free infective particles. Strain MC1061, an E. coli K-12 strain possessing wild type RecA, was susceptible to both lysis and lysogeny and was used as a control throughout.

A range of E. coli strains from ruminants, pigs, and humans and a number of representatives of other enteric bacterial genera were screened for susceptibility to lysis and/or lysogeny by 24_B::Kan. The strain groups and sources are listed in Table 1 along with the proportion of susceptible strains in each group. All of the wild-type strains were shown to be vt₂-negative by application of the PCR protocol described below for confirming lysogens after 24_B::Kan infection. Primers VT2A3' (5'-TCTGTTCAGAAACGCTGC-3') and VT2A5' (5'-TACTGTGCCTGTTACTGG-3') were designed from the published sequence of the 933W VT2 phage (GenBank accession number NC_000924) (9) to amplify the vt₂A subunit gene. All 24_B::Kan-susceptible strains were shown to be sensitive to kanamycin (50 µg ml¹) and lacked detectable norfloxacin or UV-inducible prophages.

View this table: [in this window] [in a new window]   TABLE 1.   Susceptibility of Enterobacteriaceae hosts to infection with bacteriophage 24B::Kan

To test the ability of 24_B::Kan to infect wild-type strains, cultures were grown in LB broth containing 0.01 M CaCl₂ to mid-exponential phase (optical density at 600 nm, ca. 0.5). Phage suspensions were added to a final concentration of 10⁷ PFU ml¹. Infection mixtures were incubated at 37°C with shaking at 120 rpm. Duplicate samples (100 µl) were taken after 2 h and spread on LB agar containing 50 µg of kanamycin ml¹ and incubated overnight at 37°C to select for putative lysogens. Control infections in which bacteriophage was omitted were conducted in parallel to confirm that kanamycin resistance was dependent on phage infection. Susceptibility of the strains to lytic infection by 24_B::Kan was determined by conventional plaque assay.

Of the total 113 strains tested, 30 of the 103 E. coli strains and all 4 of the Shigella strains were susceptible to lysogenic infection by 24_B::Kan, indicated by growth in the presence of kanamycin after infection. In all cases, lysogens were confirmed by detection of the inactivated vt₂A subunit gene (vt₂A::aph3) in the kanamycin-resistant colonies by PCR amplification. The primers VT2A3' and VT2A5' were used to amplify the vt₂A::aph3 gene from bacterial colonies and genomic DNA preparations from putative lysogens, to yield a 2.2-kb product in all cases. The Taq polymerase (MBI Fermentas) system was used according to the manufacturer's instructions in the presence of 1.5 mM MgCl₂. Cycling conditions were comprised of a 94°C denaturation step (4 min), a 56°C annealing step (30 s), and a 72°C extension step (2 min 45 s) for 35 cycles with the GenAmp PCR System 2400 (Perkin-Elmer). PCR products were visualized by gel electrophoresis (0.75% agarose containing 0.4 µg of ethidium bromide ml¹).

Lysogens were induced to release infective phage particles by exposing mid-exponential-phase cultures of representative kanamycin-resistant colonies to norfloxacin (1 µg ml¹) (Sigma) (4) for 1 h or to UV light (256 nm) for 40 s. Induced cultures were allowed to recover by subculture (1 ml) in fresh LB broth containing CaCl₂ (0.01 M) (2 h). Released phage particles were detected by the indirect plaque assay with DM1187-rif described above. These data are summarized in Table 1.

E. coli strains from both animal and human sources were susceptible to lysogenic infection by the phage, and this included a large number of strains isolated from the rumen. The other strains of Enterobacteriaceae species studied were not susceptible to the phage, with the exception of the Shigella sonnei and Shigella flexneri strains, whose susceptibility was not unexpected in view of their relationships to VTEC (2). Of the seven E. coli K-12 strains tested, six were susceptible to both lysogenic and lytic infection; the expected exception was strain DM1187, which is the RecA mutant susceptible only to lytic infection.

The precise sources and serotypes of the wild-type strains susceptible to 24_B::Kan are shown in Table 2. The most important feature of these data is the range of E. coli serotypes represented, including smooth strains with intact lipopolysaccharide. It has been suggested previously that VT phages can infect only rough strains of E. coli and S. sonnei (13), inferring that the phage receptor(s) is masked by lipopolysaccharide O side chains. This can now be refuted, since a smooth strain, F172, was susceptible to lytic infection (Table 2). Although the rough E. coli K-12 strains were very susceptible to both lysis and lysogeny by this VT phage, one smooth strain (serotype O107) was equally susceptible to both. Schmidt et al. (11) studied the host range of a different VT phage using a chloramphenicol resistance gene insert to inactivate vt and also found that VT genes could potentially be disseminated to different E. coli strains. However, their study examined only human isolates and a limited range of serotypes. The overwhelming preference for lysogeny rather than lysis among susceptible E. coli isolates is, nevertheless, a feature of both the data reported here and those of Schmidt and coworkers (11), with the exception of strain F172.

View this table: [in this window] [in a new window]   TABLE 2.   Description of non-K-12 E. coli and Shigella strains found to be susceptible to lysogeny by bacteriophage 24B::Kana

VT phage host range is an important indicator of the potential for vt gene transfer in clinical and agricultural environments. The VT phage used here exhibited a broad host range among E. coli and Shigella isolates, but susceptibility, i.e., the number of lysogens generated within 2 h, varied between strains (data not shown). This is evident because an enrichment step was not used following phage infection. While the reasons for this are not clear, it suggests that lysogen formation, and not just production of an observable plaque, is an important indicator of infectivity and should be an integral part of future studies on the epidemiology of temperate bacteriophages. The recA441 mutation carried by E. coli DM1187 provides a convenient tool to enable the detection of every infective particle of such lambdoid phages, including those that would otherwise be destined for lysogeny.