Multilocus Characterization Scheme for Shiga Toxin-Encoding Bacteriophages

Abstract

Shiga toxin-producing Escherichia coli (STEC) strains are food-borne pathogens whose ability to produce Shiga toxin (Stx) is due to integration of Stx-encoding lambdoid bacteriophages. These Stx phages are both genetically and morphologically heterogeneous, and here we report the design and validation of a PCR-based multilocus typing scheme. PCR primer sets were designed for database variants of a range of key lambdoid bacteriophage genes and applied to control phages and 70 stx ؉ phage preparations induced from a collection of STEC isolates. The genetic diversity residing within these populations could be described, and observations were made on the heterogeneity of individual gene targets, including the unexpected predominance of shorttailed phages with a highly conserved tail spike protein gene. Purified Stx phages can be profiled using this scheme, and the lambdoid phage-borne genes in induced STEC preparations can be identified as well as those residing in the noninducible prophage complement. The ultimate goal is to enable robust and realistically applicable epidemiological studies of Stx phages and their traits. The impact of Stx phage on STEC epidemiology is currently unknown.

Isolation and Identification of an Enterobacter cloacae Strain Producing a Novel Subtype of Shiga Toxin Type 1

“…The local public health laboratory, in turn, submitted the broth and three suspected STEC isolates to the MDL for STEC confirmation and Stx typing. The suspected STEC isolates were negative for stx 1 and stx 2 , whereas the broth tested positive for stx 1 by realtime PCR. Testing of the broth by the Vero cell assay demonstrated typical Stx cytotoxicity.…”

“…The bacterial isolate was designated M12X01451. Realtime PCR testing of M12X01451 consistently produced late cycle threshold (C T ) values (ϳ32 cycles) for stx 1 . The culture supernatant from M12X01451 produced typical Stx cytotoxicity, and the endpoint of cytotoxicity was titrated at a 1:10,000 dilution.…”

“…Primers designed from the flanking sequences were used to amplify and sequence a 2,259-bp fragment encompassing the entire stx 1 coding region of M12X01451 as well as the immediate 5= and 3= nucleotide sequences flanking stx 1 . The M12X01451 holotoxin gene sequence is 1,227 bp in length and contains a 9-bp intervening sequence between stxA 1 and stxB 1 . Comparison of the M12X01451 holotoxin gene sequence with other stx 1 sequences in the NCBI nr/nt database indicated a best match at 87% nucleotide sequence identity.…”

“…PCR amplifications using highly conserved internal stx 1 primer sequences were successful at low annealing temperatures and yielded a partial sequence for stx 1 of M12X01451. Based on this partial sequence, inverse nested PCR was performed to obtain sequence information flanking stx 1 . Primers designed from the flanking sequences were used to amplify and sequence a 2,259-bp fragment encompassing the entire stx 1 coding region of M12X01451 as well as the immediate 5= and 3= nucleotide sequences flanking stx 1 .…”

“…The acquisition of Stx genes has been mainly attributed to lysogeny by short-tailed lambdoid phages belonging to the family Podoviridae (1). These phages have a broad host range facilitated, in part, through the recognition of BamA, an outer membrane protein that is highly conserved among Gram-negative bacteria (2).…”