Assessing the genomic relatedness and evolutionary rates of persistent verotoxigenic Escherichia coli serotypes within a closed beef herd in Canada

Abstract

<jats:p>Verotoxigenic <jats:italic> <jats:named-content content-type="species"> <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.3093" xlink:type="simple">Escherichia coli</jats:ext-link> </jats:named-content> </jats:italic> (VTEC) are food- and water-borne pathogens associated with both sporadic illness and outbreaks of enteric disease. While it is known that cattle are reservoirs of VTEC, little is known about the genomic variation of VTEC in cattle, and whether the variation in genomes reported for human outbreak strains is consistent with individual animal or group/herd sources of infection. A previous study of VTEC prevalence identified serotypes carried persistently by three consecutive cohorts of heifers within a closed herd of cattle. This present study aimed to: (i) determine whether the genomic relatedness of bovine isolates is similar to that reported for human strains associated with single source outbreaks, (ii) estimate the rates of genome change among dominant serotypes over time within a cattle herd, and (iii) identify genomic features of serotypes associated with persistence in cattle. Illumina MiSeq genome sequencing and genotyping based on allelic and single nucleotide variations were completed, while genome change over time was measured using Bayesian evolutionary analysis sampling trees. The accessory genome, including the non-protein-encoding intergenic regions (IGRs), virulence factors, antimicrobial-resistance genes and plasmid gene content of representative persistent and sporadic cattle strains were compared using Fisher’s exact test corrected for multiple comparisons. Herd strains from serotypes O6:H34 (<jats:italic>n</jats:italic>=22), O22:H8 (<jats:italic>n</jats:italic>=30), O108:H8 (<jats:italic>n</jats:italic>=39), O139:H19 (<jats:italic>n</jats:italic>=44) and O157:H7 (<jats:italic>n</jats:italic>=106) were readily distinguishable from epidemiologically unrelated strains of the same serotype using a similarity threshold of 10 or fewer allele differences between adjacent nodes. Temporal-cohort clustering within each serotype was supported by date randomization analysis. Substitutions per site per year were consistent with previously reported values for <jats:italic> <jats:named-content content-type="species"> <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.3093" xlink:type="simple">E. coli</jats:ext-link> </jats:named-content> </jats:italic>; however, there was low branch support for these values. Acquisition of the phage-encoded Shiga toxin 2 gene in serotype O22:H8 was observed. Pan-genome analyses identified accessory regions that were more prevalent in persistent serotypes (<jats:italic>P</jats:italic>≤0.05) than in sporadic serotypes. These results suggest that VTEC serotypes from a specific cattle population are highly clonal with a similar level of relatedness as human single-source outbreak-associated strains, but changes in the genome occur gradually over time. Additionally, elements in the accessory genomes may provide a selective advantage for persistence of VTEC within cattle herds.</jats:p>

The Role of Escherichia coli Shiga Toxins in STEC Colonization of Cattle

“…Host-specific (niche adaptation) information is stored in the accessory genome [185], consistent with findings that STEC strains colonizing the gastrointestinal tract of cattle persistently share similar sets of accessory genes [124,165]. In turn, pre-challenge with a STEC O22:H8 strain, a serotype which has been associated with several habitats [165] but rather rarely associated with human illness, significantly reduces shedding of STEC O157:H7 by calves [234]. STEC O22:H8 did not inhibit growth of STEC O157:H7 in vitro, suggesting that strains with different levels of host adaptation may compete with more virulent strains in certain niches like at the bovine recto-anal junction [234].…”

“…In contrast, STEC spo isolates encoded more often Stx2 or ToxB, a protein involved in adherence. While possession of efa1 was more likely to be associated with a persistent than a sporadic phenotype in this study [124], efa1 was not found in any of the persistent serotypes identified in another study [165], implying that efa1 is not sufficient as biomarker for STEC per .…”

“…Even within the O157:H7 serovar only a small subset of bovine isolates has the potential to cause diseases in humans [185]. Host-specific (niche adaptation) information is stored in the accessory genome [185], consistent with findings that STEC strains colonizing the gastrointestinal tract of cattle persistently share similar sets of accessory genes [124,165]. In turn, pre-challenge with a STEC O22:H8 strain, a serotype which has been associated with several habitats [165] but rather rarely associated with human illness, significantly reduces shedding of STEC O157:H7 by calves [234].…”

“…The absence of stx2 or the entire stx2-encoding phage in isogenic mutants of one of the STEC O157:H7 strains did not significantly impact on the magnitude or duration of E. coli O157:H7 shedding [164]. Under field conditions, however, Wang et al observed the potential acquisition of the phage-encoded stx2 gene by a bovine E. coli isolate of serotype O22:H8 in a closed Canadian beef herd [165]. While the majority of such events appear to be transient in nature, carriage of stx2 in O22:H8 persisted and strains carrying stx2 became the dominant O22:H8 genotype in the cattle cohort sampled one year later [165].…”

“…Under field conditions, however, Wang et al observed the potential acquisition of the phage-encoded stx2 gene by a bovine E. coli isolate of serotype O22:H8 in a closed Canadian beef herd [165]. While the majority of such events appear to be transient in nature, carriage of stx2 in O22:H8 persisted and strains carrying stx2 became the dominant O22:H8 genotype in the cattle cohort sampled one year later [165]. This argues in favor of a selective advantage of stx-phage carriage for the respective E. coli strain in the long term.…”