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Escherichia coli O157:H7 strains harbor at least three distinct sequence types of Shiga toxin 2a-converting phages.

Yin S, Rusconi B, Sanjar F, Goswami K, Xiaoli L, Eppinger M, Dudley EG - BMC Genomics (2015)

Bottom Line: The PST2 cluster, identified in two clade 8 strains, was related to stx2a-converting phages previously identified in non-O157 Shiga-toxin producing E. coli (STEC) strains associated with a high incidence of HUS.Diversification within a phage type is mainly driven by IS629 and by a small number of SNPs.Polymorphisms between phage genomes may help explain differences in Stx2a production between strains, however our data indicates that genes encoded external to the phage affect toxin production as well.

View Article: PubMed Central - PubMed

Affiliation: Department of Food Science, The Pennsylvania State University, University Park, PA, 16802, USA.

ABSTRACT

Background: Shiga toxin-producing Escherichia coli O157:H7 is a foodborne pathogen that causes severe human diseases including hemolytic uremic syndrome (HUS). The virulence factor that mediates HUS, Shiga toxin (Stx), is encoded within the genome of a lambdoid prophage. Although draft sequences are publicly available for a large number of E. coli O157:H7 strains, the high sequence similarity of stx-converting bacteriophages with other lambdoid prophages poses challenges to accurately assess the organization and plasticity among stx-converting phages due to assembly difficulties.

Methods: To further explore genome plasticity of stx-converting prophages, we enriched phage DNA from 45 ciprofloxacin-induced cultures for subsequent 454 pyrosequencing to facilitate assembly of the complete phage genomes. In total, 22 stx2a-converting phage genomes were closed.

Results: Comparison of the genomes distinguished nine distinct phage sequence types (PSTs) delineated by variation in obtained sequences, such as single nucleotide polymorphisms (SNPs) and insertion sequence element prevalence and location. These nine PSTs formed three distinct clusters, designated as PST1, PST2 and PST3. The PST2 cluster, identified in two clade 8 strains, was related to stx2a-converting phages previously identified in non-O157 Shiga-toxin producing E. coli (STEC) strains associated with a high incidence of HUS. The PST1 cluster contained phages related to those from E. coli O157:H7 strain Sakai (lineage I, clade 1), and PST3 contained a single phage that was distinct from the rest but most related to the phage from E. coli O157:H7 strain EC4115 (lineage I/II, clade 8). Five strains carried identical stx2a-converting phages (PST1-1) integrated at the same chromosomal locus, but these strains produced different levels of Stx2.

Conclusion: The stx2a-converting phages of E. coli O157:H7 can be categorized into at least three phage types. Diversification within a phage type is mainly driven by IS629 and by a small number of SNPs. Polymorphisms between phage genomes may help explain differences in Stx2a production between strains, however our data indicates that genes encoded external to the phage affect toxin production as well.

No MeSH data available.


Related in: MedlinePlus

Host factors modulate stx2a expression levels. (a) Whole genomes of 14 O157:H7 strains with PST1-1 were aligned using Mugsy [44] and the phylogenetic tree was inferred using RaxML [45] with 100 bootstrap replicates and visualized using Figtree [88]. (b) Stx2 production was quantified by ELISA 2 h after ciprofloxacin induction. Bars labeled with different letters were significantly different (P < 0.05)
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Fig7: Host factors modulate stx2a expression levels. (a) Whole genomes of 14 O157:H7 strains with PST1-1 were aligned using Mugsy [44] and the phylogenetic tree was inferred using RaxML [45] with 100 bootstrap replicates and visualized using Figtree [88]. (b) Stx2 production was quantified by ELISA 2 h after ciprofloxacin induction. Bars labeled with different letters were significantly different (P < 0.05)

Mentions: Our analyses identified for the first time strains of E. coli O157:H7 (PA4, PA18, PA30, PA33, and PA44) carrying stx2a-converting phages (PST1-1) that were 100 % identical on the phDNA level and had the same insertion site (wrbA). These strains also carry an stx1-converting phage [28]. This provided us with the unique opportunity to use non-manipulated strains to ask whether factors apart from the stx2a-converting phage genome impact toxin production. To first investigate the genetic relatedness of the host strains, we established a phylogenomic framework derived from whole genome alignments of shared regions, excluding prophage, for all 45 E. coli O157:H7 genomes [44, 45] (Fig. 6). Strains PA4, PA18, PA30, PA33 and PA44 are phylogenetically positioned on separate branches indicating that, as expected, no two chromosomes genomes were identical. Next, we measured the Stx2a levels and stx2a expression after ciprofloxacin phage-induction (Fig. 7a). The lowest Stx2a producer was PA4, at approximately 1 ng/μg of protein, and the highest producer was PA44, at approximately 5 ng/μg of protein. PA18 produced 3.2 ng/μg, which was significantly different from the previous two strains, while toxin production from strains PA30 (4.4 ng/μg) and PA33 (4.6 ng/μg) was only different from PA4. Surprisingly, stx2a transcript levels were similar for all strains except for PA18, which was greater than 10-fold above that measured with PA4, PA30, PA33, or PA44 (Fig. 7b). This observed disconnect indicates that qPCR does not always reflect the level of Stx2 secreted by E. coli O157:H7.Fig. 6


Escherichia coli O157:H7 strains harbor at least three distinct sequence types of Shiga toxin 2a-converting phages.

Yin S, Rusconi B, Sanjar F, Goswami K, Xiaoli L, Eppinger M, Dudley EG - BMC Genomics (2015)

Host factors modulate stx2a expression levels. (a) Whole genomes of 14 O157:H7 strains with PST1-1 were aligned using Mugsy [44] and the phylogenetic tree was inferred using RaxML [45] with 100 bootstrap replicates and visualized using Figtree [88]. (b) Stx2 production was quantified by ELISA 2 h after ciprofloxacin induction. Bars labeled with different letters were significantly different (P < 0.05)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4587872&req=5

Fig7: Host factors modulate stx2a expression levels. (a) Whole genomes of 14 O157:H7 strains with PST1-1 were aligned using Mugsy [44] and the phylogenetic tree was inferred using RaxML [45] with 100 bootstrap replicates and visualized using Figtree [88]. (b) Stx2 production was quantified by ELISA 2 h after ciprofloxacin induction. Bars labeled with different letters were significantly different (P < 0.05)
Mentions: Our analyses identified for the first time strains of E. coli O157:H7 (PA4, PA18, PA30, PA33, and PA44) carrying stx2a-converting phages (PST1-1) that were 100 % identical on the phDNA level and had the same insertion site (wrbA). These strains also carry an stx1-converting phage [28]. This provided us with the unique opportunity to use non-manipulated strains to ask whether factors apart from the stx2a-converting phage genome impact toxin production. To first investigate the genetic relatedness of the host strains, we established a phylogenomic framework derived from whole genome alignments of shared regions, excluding prophage, for all 45 E. coli O157:H7 genomes [44, 45] (Fig. 6). Strains PA4, PA18, PA30, PA33 and PA44 are phylogenetically positioned on separate branches indicating that, as expected, no two chromosomes genomes were identical. Next, we measured the Stx2a levels and stx2a expression after ciprofloxacin phage-induction (Fig. 7a). The lowest Stx2a producer was PA4, at approximately 1 ng/μg of protein, and the highest producer was PA44, at approximately 5 ng/μg of protein. PA18 produced 3.2 ng/μg, which was significantly different from the previous two strains, while toxin production from strains PA30 (4.4 ng/μg) and PA33 (4.6 ng/μg) was only different from PA4. Surprisingly, stx2a transcript levels were similar for all strains except for PA18, which was greater than 10-fold above that measured with PA4, PA30, PA33, or PA44 (Fig. 7b). This observed disconnect indicates that qPCR does not always reflect the level of Stx2 secreted by E. coli O157:H7.Fig. 6

Bottom Line: The PST2 cluster, identified in two clade 8 strains, was related to stx2a-converting phages previously identified in non-O157 Shiga-toxin producing E. coli (STEC) strains associated with a high incidence of HUS.Diversification within a phage type is mainly driven by IS629 and by a small number of SNPs.Polymorphisms between phage genomes may help explain differences in Stx2a production between strains, however our data indicates that genes encoded external to the phage affect toxin production as well.

View Article: PubMed Central - PubMed

Affiliation: Department of Food Science, The Pennsylvania State University, University Park, PA, 16802, USA.

ABSTRACT

Background: Shiga toxin-producing Escherichia coli O157:H7 is a foodborne pathogen that causes severe human diseases including hemolytic uremic syndrome (HUS). The virulence factor that mediates HUS, Shiga toxin (Stx), is encoded within the genome of a lambdoid prophage. Although draft sequences are publicly available for a large number of E. coli O157:H7 strains, the high sequence similarity of stx-converting bacteriophages with other lambdoid prophages poses challenges to accurately assess the organization and plasticity among stx-converting phages due to assembly difficulties.

Methods: To further explore genome plasticity of stx-converting prophages, we enriched phage DNA from 45 ciprofloxacin-induced cultures for subsequent 454 pyrosequencing to facilitate assembly of the complete phage genomes. In total, 22 stx2a-converting phage genomes were closed.

Results: Comparison of the genomes distinguished nine distinct phage sequence types (PSTs) delineated by variation in obtained sequences, such as single nucleotide polymorphisms (SNPs) and insertion sequence element prevalence and location. These nine PSTs formed three distinct clusters, designated as PST1, PST2 and PST3. The PST2 cluster, identified in two clade 8 strains, was related to stx2a-converting phages previously identified in non-O157 Shiga-toxin producing E. coli (STEC) strains associated with a high incidence of HUS. The PST1 cluster contained phages related to those from E. coli O157:H7 strain Sakai (lineage I, clade 1), and PST3 contained a single phage that was distinct from the rest but most related to the phage from E. coli O157:H7 strain EC4115 (lineage I/II, clade 8). Five strains carried identical stx2a-converting phages (PST1-1) integrated at the same chromosomal locus, but these strains produced different levels of Stx2.

Conclusion: The stx2a-converting phages of E. coli O157:H7 can be categorized into at least three phage types. Diversification within a phage type is mainly driven by IS629 and by a small number of SNPs. Polymorphisms between phage genomes may help explain differences in Stx2a production between strains, however our data indicates that genes encoded external to the phage affect toxin production as well.

No MeSH data available.


Related in: MedlinePlus