Detection limits of the DH and TSPE-UH suspension array formats.

a<p>Values displayed represent the lowest DNA concentration at which 95% of the positive samples are detected, as calculated by using probit analysis. ND = not determined.</p

Typical results from DH and TSPE-UH suspension microarrays detecting select pathogens.

<p>Two 17-plex bead arrays were developed for the detection of <i>B. anthracis</i> (Ba), <i>F. tularensis</i> (Ft), <i>Y. pestis</i> (Yp), <i>C. burnetii</i> (Cb) and an internal control for DNA extraction and microarray detection (Bt). The microarrays were based on (<b>A</b>) direct hybridization (DH), or (<b>B</b>) target specific primer extension combined with universal microarray hybridization (TSPE-UH) assay formats. Both microarrays make use of identical amplification products from a 16-plex asymmetric PCR. Mean fluorescence intensity (MFI) is displayed for the different probes that are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031958#pone-0031958-t001" target="_blank">Table 1</a>.</p

DH suspension microarray measurement showing minor cross-reactivity.

<p>Three <i>Y. pestis</i> strains (Yp) and the no template control (NTC) are shown from a DH measurement. Probe <i>isf</i> showed a minor signal when a high load of <i>Y. pestis</i> genomic DNA was amplified.</p

Oligonucleotides used for amplification and labeling of signature sequences and as fixed probes.

a<p>Excess primer = X, Limiting primer = L.</p>b<p><i>F. tularensis</i> subspecies <i>tularensis</i> yields amplicon of 307 bp, subspecies <i>novicida</i> and <i>mediasiatica</i> of 451 bp.</p

Detection of mixed pathogens by using DH and TSPE-UH suspension microarrays.

<p>Genomic DNA from <i>B. anthracis</i> (Ba), <i>F. tularensis</i> (Ft), <i>Y. pestis</i> (Yp), <i>C. burnetii</i> (Cb) was mixed in different ratios and measured by using DH (<b>A</b>) and TSPE-UH (<b>B</b>) microarrays. Mean fluorescence intensity (MFI) is displayed for the different pathogen-specific probes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031958#pone-0031958-t001" target="_blank">Table 1</a>). The detection of one pathogen is not impeded by the detection of the other targeted pathogens.</p

Wastewater samples.

<p>HCI = Health Care Institution, WWTP = Wastewater Treatment Plant</p><p>^aEffluents were obtained three (^*) or four (^ǂ) times during 2012.</p><p>Underlined years indicate that influents and effluents from the same year and location were sampled at the same time-point.</p><p>Wastewater samples.</p

Surface water samples.

<p>^aA total of 113 samples were taken at 30 locations scattered over eight different regions (group A and group H contained different water bodies, but were located in the same regional area). For each group, the number of samples analysed per location is calculated by dividing the no. of samples by the no. of locations; this number also equals the number of different sampling dates per site.</p><p>Surface water samples.</p

Characteristics of ESBL-producing <i>E</i>. <i>coli</i> isolates from wastewater chains.

<p>* Am = ampicillin</p><p>Cx = cefotaxime</p><p>Cz = ceftazidime</p><p>Te = tetracyclin</p><p>St = streptomycin</p><p>Ci = ciprofloxacine</p><p>Na = nalidixic acid</p><p>Su = sulfamethoxazole</p><p>Tr = trimethoprim</p><p>Ch = chloramphenicol.</p><p>MICs were determined with Etests (Cz, Na, Su, Ch) and microbroth dilution (remainder of antimicrobials), resistance defined as MIC greater than or equal to the epidemiological cut-off (EUCAST).</p><p>Indicated in bold are pheno-/genotypes present in HCI and municipal wastewater.</p><p>Characteristics of ESBL-producing <i>E</i>. <i>coli</i> isolates from wastewater chains.</p

Resistant and multi-drug resistant <i>E</i>. <i>coli</i> in surface water (A) and wastewater (B).

<p>For each group of isolates from surface water (A) and wastewater (B), percentages of isolates resistant to 0, or 1 to 7 different antimicrobial classes are shown. On the x-axis, indicated between brackets (n = x;y;z) are the number of isolates (x), the number of samples (y), and the number of sample locations (z). MDR = multidrug resistant, i.e. resistant to three or more different classes of antimicrobials, AMR = antimicrobial resistant, i.e. resistant to at least one class of antimicrobials. For airport wastewater results from influent and effluent were combined because of small sample sizes. **P<0.01 relative to surface water values, Pearson Chi-Square Test.</p

AMR resistance and relative distribution of resistance types among <i>E</i>. <i>coli</i> from different water sources and livestock

<p>Indicated are (A) the percentages of <i>E</i>. <i>coli</i> with the indicated type of resistance, proportional to the total number of <i>E</i>. <i>coli</i> (n_ec) obtained from the indicated water sources and (B) the proportion of the indicated types of resistance, relative to the total number of resistances (n_r) in the respective <i>E</i>. <i>coli</i> populations.</p><p>**P<0.01 and</p><p>*P<0.05 relative to surface water values, Pearson Chi Square Test.</p><p>mWWTP = municipal wastewater treatment plant</p><p>ww = wastewater</p><p>HCI-health care institution.</p><p>^a The sum of all percentages does not add-up to the total percentage of AMR isolates because of the existence of multidrug resistant isolates.</p><p>^bCalculated from the numbers of <i>E</i>. <i>coli</i> isolates and the percentages of <i>E</i>. <i>coli</i> isolates resistant to the antimicrobials under study reported in MARAN 2012 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127752#pone.0127752.ref034" target="_blank">34</a>].</p><p>AMR resistance and relative distribution of resistance types among <i>E</i>. <i>coli</i> from different water sources and livestock</p