Analysis of Escherichia coli O157 strains in cattle and humans between Scotland and England & Wales: implications for human health

For the last two decades, the human infection frequency of Escherichia coli O157 (O157) in Scotland has been 2.5-fold higher than in England and Wales. Results from national cattle surveys conducted in Scotland and England and Wales in 2014/2015 were combined with data on reported human clinical cases from the same time frame to determine if strain differences in national populations of O157 in cattle could be associated with higher human infection rates in Scotland. Shiga toxin subtype (Stx) and phage type (PT) were examined within and between host (cattle vs human) and nation (Scotland vs England and Wales). For a subset of the strains, whole genome sequencing (WGS) provided further insights into geographical and host association. All three major O157 lineages (I, II, I/II) and most sub-lineages (Ia, Ib, Ic, IIa, IIb, IIc) were represented in cattle and humans in both nations. While the relative contribution of different reservoir hosts to human infection is unknown, WGS analysis indicated that the majority of O157 diversity in human cases was captured by isolates from cattle. Despite comparable cattle O157 prevalence between nations, strain types were localized. PT21/28 (sub-lineage Ic, Stx2a+) was significantly more prevalent in Scottish cattle [odds ratio (OR) 8.7 (2.3–33.7; P<0.001] and humans [OR 2.2 (1.5–3.2); P<0.001]. In England and Wales, cattle had a significantly higher association with sub-lineage IIa strains [PT54, Stx2c; OR 5.6 (1.27–33.3); P=0.011] while humans were significantly more closely associated with sub-lineage IIb [PT8, Stx1 and Stx2c; OR 29 (4.9–1161); P<0.001]. Therefore, cattle farms in Scotland were more likely to harbour Stx2a+O157 strains compared to farms in E and W (P<0.001). There was evidence of limited cattle strain migration between nations and clinical isolates from one nation were more similar to cattle isolates from the same nation, with sub-lineage Ic (mainly PT21/28) exhibiting clear national association and evidence of local transmission in Scotland. While we propose the higher rate of O157 clinical cases in Scotland, compared to England and Wales, is a consequence of the nationally higher level of Stx2a+O157 strains in Scottish cattle, we discuss the multiple additional factors that may also contribute to the different infection rates between these nations.

Historical data was obtained from annual reports from Public Health England (PHE) [1], The Scottish E. coli / STEC Reference Laboratory (SERL) [2] and Public Health Scotland (PHS, formerly Health Protection Scotland (HPS)) [3] for the total number of reported human clinical cases (Fig. S1.1A) and the distribution of phage types (PT) from 1998 to 2019 (Fig. S1.1B).We chose to start with the year 1998 as there was a large outbreak in Central Scotland in 1996/1997 which inflated the case numbers.
The yearly count data includes outbreak and travel-related cases.The case data was analyzed using Poisson regression analysis of the number of human reported clinical cases in Scotland and England & Wales 1998-2019 adjusted for population size (mid-year population size [4]).The model was fit using a generalised linear model with a Poisson distribution fitted to count data, offset by the logarithm of population size, to identify differences in the rates of O157 infection across time (year: 1998-2019; p=0.001) and location (Scotland vs England & Wales; p<0.001) (Table S1.1).The interaction was not significant so it was removed from the model (p=0.9626).The overall rate of O157 is declining in the UK (Fig. S1.1A).There was a significant difference between the rate of O157 in Scotland    .This includes all samples submitted to SERL, PHE and PHS over a given calendar year .Travelrelated cases and outbreak cases are included.The graph demonstrates

Proportion of cases for different Phage Types
• the high proportion of cases of PT21/28 in Scotland, especially between 1997 (when it replaced PT2) to 2011 (when the proportion of PT8 cases increased to the same level of PT21/28).• Strain replacement of PT2 by PT21/28 and the decline of PT21/28 and increase of PT8 in both Scotland and England & Wales

Spatial Regions of Scotland, England & Wales
In Scotland, in previous publications we have defined

Outbreaks
Five SNP single linkage clusters (SLC) have been used operationally in the UK to define isolates that are likely to be epidemiologically linked [1].S3.1) contained greater than 5 cases and were investigated epidemiologically as potential outbreaks over the time frame of this study.Three of these clusters were associated with the consumption of bagged salad of domestic origin, two in lineage IIc (Fig. S3.1) and one in lineage IIb (Fig. S3.2).Four outbreaks occurred in E&W involving lineage Ic strains in the study period (Fig. S3.3).This included OB4 associated with a butcher's shop and linked back to a cattle farm in the North East [2], OB5 associated with slaw consumption [3], OB6 associated with a private water supply in the North West [4], and OB7 associated with the consumption of raw drinking milk [5] sourced from the South West of England.In all four outbreaks the exposure could be linked to food, animal, or environmental exposure.Supplementary material 4: Data Tables for Figure 1 in main manuscript Table S4.1 A&B contain the data associated with Fig. 1 (4.0 (3.6-4.3)) and England and Wales (1.6 (1.5-1.7)).Analyses were carried out using Proc Glimmix in SAS version 9.4 (SAS Institute Inc., Cary, NC)

Fig. S1 .
Fig. S1.1B shows the proportion of cases of the predominant phage types (PT) In England & Wales and Scotland.This includes all samples submitted to SERL, PHE and PHS over a given calendar year.Travelrelated cases and outbreak cases are included.The graph demonstrates• the high proportion of cases of PT21/28 in Scotland, especially between 1997 (when it replaced PT2) to 2011 (when the proportion of PT8 cases increased to the same level of PT21/28).• Strain replacement of PT2 by PT21/28 and the decline of PT21/28 and increase of PT8 in both Scotland and England & Wales

Figure S1 .
Figure S1.1BProportion of cases of the predominant phage types (PT) in England & Wales (dashed lines) and Scotland (solid lines).

Appendix 2 :
Fig. S2.1 shows how Lineage, PT, and stx subtype are associated within this study.Diagram was constructed using Interactive Tree Of Life (iTOL) program[1]  and included all clinical (n=684) and cattle (n=113) isolates sequenced within this study.

Figure S2. 1
Figure S2.1 Association among Lineage (ring 1), PT (ring 2), and stx subtype (ring 3-5) for the isolates in this study.Rings are defined from the inner circle to out.
Fig. S3.1 Maximum likelihood phylogenies of clinical and cattle isolates from lineage IIC.Taxa are labelled 'cattle' or '*' for clinical Fig. S3.2 Maximum likelihood phylogenies of clinical and cattle isolates from lineage IIB.Taxa are labelled 'cattle' or '*' for clinical Fig. S3.3 Maximum likelihood phylogenies of clinical and cattle isolates from lineage IC.Taxa are labelled 'cattle' or '*' for clinical

.1A Rate per 100,000 population of reported clinical E. coli O157 cases in Scotland and England & Wales between 1998 and 2019.
From the 684 clinical cases from England, Wales and Scotland, there were 82 clusters (2 or more isolates) and 70 (85%) of these contained isolates from only one of the two regions (Scotland or England & Wales), indicating that exposures were generally restricted at a national level.The picture is different if larger clusters or outbreaks are examined with 57% (4/7) of having cases in both England & Wales and Scotland.The seven clusters (Table

: Data Tables: Strain composition Table S5.1 Complete list of all possible stx subtypes for each phage type. There is a tick (✓) in the cell if the strain was found in cattle or human clinical cases from Scotland or England or Wales.
(Pearce et al., 2009;Henry et al., 2017)ts of Phage types and presence/absence of E. coli O157 is expressed for the 6 Animal health Districts (AHDs) (Fig.S1.2A) as done historically(Pearce et al., 2009;Henry et al., 2017)including the following: Highland, Islands, North East, Central, South East, South West.In England & Wales spatial regions were defined using the Nomenclature of Units for Territorial Statistics (NUTS) (Figure S4.1B).NUTS 1 regions for England & Wales include: North east, North West, Yorkshire, East Midlands, West Midlands, East of England, London (no data), South east, South West, West.Phage types (PT) were grouped as follows: PT1, PT32, PT4, PT2, PT54, PT8, PT21/28 and PT Other (other includes the following PTs: 14, 31, 34, RDNC).Spatial regions were defined to ensure at least 5 farms in each region to preserve confidentiality.European Parliament, Council of the European Union.Regulation (EC) No 1059/2003 of the European Parliament and of the Council of 26 May 2003 on the establishment of a common classification of territorial units for statistics (NUTS).Official Journal of the European Union L154 2003; 46:1 Table S4.1.Data used in Figure 1 of the main manuscript for (A) Scotland and (B) England & Wales.