Additional file 1: S.01. of Rationale and design of ASPIRE-ICU: a prospective cohort study on the incidence and predictors of Staphylococcus aureus and Pseudomonas aeruginosa pneumonia in the ICU

Complete list of objectives and endpoints. Table S 02. Schedule of procedures. S.03. Complete definition of study endpoints. (DOCX 29 kb

<i>NetB</i> toxin transcription is not influenced by DON.

<p>Transcription level of <i>netB</i> toxin was analysed by qRT-PCR of <i>C. perfringens</i> strain 56 RNA samples collected from <i>in vitro</i> culture material in the mid (after 3 h incubation) and late logarithmic (after 6 h incubation) growth phase. <i>C. perfringens</i> strain 56 was grown in absence or presence (0.2, 2, 20 µg/mL) of DON. The results for the <i>netB</i> gene transcription were normalized to the <i>rpoA</i> gene transcription. Results are presented as the mean value of three biological replicates. Error bars represent SD. There is no significant difference between the different test conditions.</p

Deoxynivalenol predisposes for <i>C. perfringens</i> induced necrotic enteritis.

<p>DON decreased villus height and reduced transepithelial electrical resistance (1), leading to a decreased absorption and digestion of dietary nutrients; and an increased intestinal barrier permeability, respectively. Taken together with an increased intestinal protein level, these results suggest an impaired nutrient uptake (2) and leakage of plasma amino acids (3) into the intestinal lumen, providing the necessary growth substrate for <i>C. pefringens</i> proliferation (4). Proliferation of virulent (<i>netB</i> positive) <i>C. perfringens</i> induces necrotic enteritis (5).</p

Protein concentration in intestinal content is significantly increased in duodenum of chickens fed a DON-contaminated diet.

<p>Percentage crude protein per dry matter of the intestinal content was determined by the Kjeldahl method. Results are presented as the mean protein level of 27 samples per group per intestinal segment. Error bars represent SD. ^(*) significantly different (<i>P</i><0.05) within one intestinal segment.</p

Lesion scores of individual broiler chickens challenged with <i>C. perfringens</i>.

<p>Chickens were fed either a control or DON-contaminated diet and subsequently challenged with <i>C. perfringens</i> strain 56. The solid bars represent the average lesion score in each group. Error bars represent SEM. Intestinal lesions in the small intestine (duodenum to ileum) were scored as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108775#pone.0108775-Keyburn1" target="_blank">[7]</a>; 0 no gross lesions; 2 small focal necrosis or ulceration (one to five foci); 3 focal necrosis or ulceration (six to 15 foci); 4 focal necrosis or ulceration (16 or more foci); 5 patches of necrosis 2 to 3 cm long; 6 diffuse necrosis typical field cases. The score 1 used for congested intestinal mucosa was not applied here because of difficulties in scoring this characteristic objectively, and due to the lack of scientific documentation of an association between “congested intestinal mucosa” and necrotic enteritis. Birds with lesion scores of 2 or more were classified as NE positive.</p

Effect of DON on villus height and crypt depth measurements.

<p>Analysis was based on 9 animals per treatment, and the mean of 5 to 15 measurements per segment per animal was calculated; data are presented as weighted mean ± SEM.</p>(*)<p>significantly different (P<0.05).</p><p>Effect of DON on villus height and crypt depth measurements.</p

No impact of DON on <i>in vitro</i> growth of <i>C. perfringens</i>.

<p><i>C. perfringens</i> strains 6 (a) and 56 (b) were grown in TGY broth medium containing 0, 0.2, 2 or 20 µg DON/mL. Samples were taken at 0, 2, 3, 4, 5, 6, 7, 8 and 24 h after inoculation with an overnight culture of <i>C. perfringens.</i> The number of colony forming units (cfu) per mL was determined by bacterial plating of 10-fold dilutions. Results are presented as the mean cfu/mL. There is no significant difference between the different test conditions.</p

Experimental groups and impact of DON on the number of chickens affected by necrotic enteritis (NE) and bodyweight (BW) gain.

<p>Four experimental groups were included, of which each experimental group consisted of 3 cages of 30 chickens. After a feeding period of 3 weeks chickens were euthanized.</p>(1)<p>Cp: <i>C. perfringens</i> challenge strain 56; DON: deoxynivalenol challenge.</p>(2)<p>Results bodyweight (BW) gain based on ten animals per group in triplicate.</p>(3)<p>Since the mean NE lesion score in groups DON alone and negative control was zero, both groups were excluded from statistical analysis with respect to macroscopic NE lesion scoring.</p>a–b<p>significantly different within one column (<i>P</i><0.05). All data are presented as mean ± standard deviation.</p><p>Experimental groups and impact of DON on the number of chickens affected by necrotic enteritis (NE) and bodyweight (BW) gain.</p

Additional file 1 of Safety, efficacy, and pharmacokinetics of gremubamab (MEDI3902), an anti-Pseudomonas aeruginosa bispecific human monoclonal antibody, in P. aeruginosa-colonised, mechanically ventilated intensive care unit patients: a randomised controlled trial

Additional file 1: Supplementary Methods, Results, Tables and Figures