Assignment of faecal and systemic <i>E. coli</i> to phylogenetic groups.

<p>216 faecal <i>E. coli</i> and 35 systemic <i>E. coli</i> from vital organs were typed using the Clermont <i>et al</i> triplex PCR and assigned to 1 of 5 phylogenetic groups. Those isolates which showed no amplification of any of the 3 targets, yet confirmed to be <i>E. coli</i> were assigned to subgroup A0. Group D was the most frequently detected phylogenetic group among faecal population, while A0 (untypable) was the most common group among systemic isolates. B2 and D have been previously associated with more pathogenic <i>E. coli</i>, however they only accounted for 28.57% of systemic isolates in this study.</p

VAG profile diversity for all flocks.

<p><i>a</i>) Shows the VAG profiles identified at t = 0 (arrival of chicks). Profiles consisting of 4 VAGs were the most diverse, with differences in iron acquisition genes being the most abundant, while profiles 0010101110 (<i>irp2</i>⁺, <i>papC⁺</i>, <i>va</i>t⁺,<i>cvi</i>⁺, <i>sitA</i>⁺) and 0011101010 (<i>irp2</i>⁺, <i>iucD</i>⁺, <i>papC</i>⁺, <i>va</i>t⁺, <i>sitA</i>⁺) both with 5 VAGs were the most common profile identified <i>b</i>) Shows the VAG profiles identified at t  =  week 5. VAG profile diversity had declined over time. Most diversity was detected with the possession of 3 VAGs. No isolates carried more than 5 VAGs <i>c</i>) Comparison of total number of VAGs carried by individually tested <i>E. coli</i> at t = 0 and 5. Profile 206 (0000000000) excluded from both graphs. Not all profiles were represented in all four cycles.</p

Observed faecal and systemic <i>E. coli</i> MLST Sequence types categorised by VAG carriage.

<p>(n) <i> = </i> ST observation frequency. All faecal <i>E. coli</i> belonged to newly identified sequence types (ST) excluding ST-352. Interestingly, all ST-352 isolates harboured more than 5 VAGs with the following profiles:1) <i>astA</i>⁺, <i>irp2</i>⁺, <i>papC</i>⁺, <i>iucD</i>⁺, <i>vat</i>⁺, <i>cvi</i>⁺, <i>sitA</i>⁺ 2) <i>iss</i>⁺, <i>irp2</i>⁺, <i>papC</i>⁺, <i>iucD</i>⁺, <i>vat</i>⁺, <i>cvi</i>⁺, <i>sitA</i>⁺ 3) <i>iss</i>⁺, <i>irp2</i>⁺, <i>papC</i>⁺, <i>iucD</i>⁺, <i>vat</i>⁺, <i>cvi</i>⁺, <i>sitA</i>⁺ and they did not group with other <i>E. coli</i> in the online database. Systemic <i>E. coli</i> analysis identified 3 ST–117 and 4 ST- 2999 isolates; however ST-2998 and ST-3000 did not cluster with the other two STs in this category.</p

Dendrogram constructed using DICE for systemic <i>E.</i><i>coli</i>. (tolerance 5%)

<p>(minimum height >0.0%, minimum surface >0.0%)(0.0–100% coefficient) for XbaI PFGE. A dendrogram showing the strain diversity amongst systemic E. <i>coli</i> harbouring APEC VAGs constructed using BioNumerics software by unweighted pair group method with Arithmetic mean. The dendrogram also shows; phylogenetic group (P) (green  =  D; red  =  A; yellow  =  B2; blue  =  B1), isolate (I), organ and age of bird at isolation (H  =  heart: K  =  kidney: Li  =  liver: Lu  =  lung; S  =  spleen), MLST sequence type (ST) and VAG profiles. The dendrogram shows the clustering of ST 117 and 2999 isolates (excluding 601) which by PFGE analysis are ∼60% different from other isolates. Several ST 3004 were identified and these potentially show the acquisition of 2 Iron acquisition genes (<i>irp2</i> and <i>iucD</i>) while other ST 3004 isolates have no VAGs (isolates 579 and 583).</p

Average percentage of pAPEC with respect to time.

<p>At weekly intervals the average percentage of potential APEC, defined by the carriage of ≥5 VAGs, from the total faecal <i>E.coli</i> population was calculated. At each time point, 160 faecal <i>E. coli</i> were assessed. 95% upper confidence interval error bars shown. Overall, there is a general decline with time; the average detection frequency at placement of chicks (week 0) was 24.05% and only 1% by week 5.</p

Simpson's diversity index for VAG profile diversity through time.

<p>Simpson's diversity index (D) was used to compare VAG profile diversity through time in the second flock cycles of farm 1 (F1) and farm 2 (F2). Overall, profile diversity decreases with time, with a peak at week 3.</p

Comparison of faecal and systemic <i>E.</i><i>coli</i> VAG carriage.

<p>Upper and lower bound 95% confidence intervals indicate statistically significant differences between VAG carriages by the two populations. Fisher's exact test indicates that <i>irp2, papC, iucD, cvi, sitA</i> and <i>ibeA</i> are significantly more associated with systemic <i>E. coli</i> (p<0.05) denoted in figure by *.</p

The main phenotypic differences as determined by Omnilog analysis of 7h1h and RM4018.+represents growth and – represents no growth of the isolate on the omnilog plate.

<p>The main phenotypic differences as determined by Omnilog analysis of 7h1h and RM4018.+represents growth and – represents no growth of the isolate on the omnilog plate.</p

Subsystems identified by RAST in isolates 7h1h and RM4018.

<p>Subsystems identified by RAST in isolates 7h1h and RM4018.</p

Example of metabolic variation between sequenced isolates.

<p>Omnilog output chart showing the growth curves of 7h1h (blue) and RM4018 (red) using L-asparagine and L-glutamic acid as carbon sources over a time period of 72 hours. Each isolate was tested in duplicate, hence 7h1ha, 7h1hb, RM4018a and RM4018b. The red and blue lines along the bottom of the charts include negative controls for each isolate.</p