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

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

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

Bacterial concentrations in surface waters (A) and waste water (B).

<p>For purpose of comparison with concentrations of ESBL-producing <i>E</i>. <i>coli</i> which were only determined in samples from 2010 onwards, in (A), samples taken prior to 2010 are indicated in black, and samples taken from 2010 onwards are indicated in white. In (B), grey symbols indicate HCI wastewater, black symbols indicate WWTP influents, and white symbols indicate WWTP effluents. Horizontal bars indicate median values. Samples with concentrations below the detection limit (<i>E</i>. <i>coli</i>: <10 cfu/ml, ESBL-<i>E</i>. <i>coli</i>:.<1.5 cfu/ml) are represented by symbols on the x-axis.</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

Numbers of characterized ESBL-producing <i>E</i>. <i>coli</i> isolates per matrix and farm type.

<p>* Indicated between brackets are the numbers of isolates that were not characterized using MLST.</p><p>Numbers of characterized ESBL-producing <i>E</i>. <i>coli</i> isolates per matrix and farm type.</p

Relationship between ESBL-producing variants from different matrices.

<p>Indicated are the proportions of isolates from the different environmental matrices, with identical counterparts in manure and rinse water and/or other environmental matrices at the same farm, based on phylogenetic group, ESBL-genotype, ABR profile and ST. White bars represent broiler farms, black bars represent laying hen farms, and hatched bars represent the results for all farms combined.</p

ESBL-producing <i>E</i>. <i>coli</i> variants on laying hen farms.

<p>Maximum parsimony trees constructed based on the concatenated sequences of the seven MLST alleles, using Bionumerics 7.1 software. Node sizes reflect the number of isolates per ST and node colours represent different matrices. Additionally indicated are the phylogenetic group, ESBL-genotype and ABR profiles of isolates in every node (i.e., ST). Note that per sample maximally one isolate of each variant was included, and that multiple isolates of a specific variant reflects detection in multiple samples. ABR profiles represent antibiotics to which resistance was observed additionally to 3rd generation cephalosporins. In between brackets are antibiotics with MICs just above and just below epidemiological cut-off values (i.e. with a maximal 2-fold difference) among isolates with the same ST and/or ESBL genotype. Sx = sulfamethoxazole, Tm = trimethoprim; Te = tetracycline, Ci = ciprofloxacin, Na = nalidixic acid, St = streptomycin, Ge = gentamycin, Ax = amoxicillin+clavulanic acid, Ch = chloramphenicol; u.i.d. = unidentified.</p

ESBL-producing <i>E</i>. <i>coli</i> and <i>E</i>. <i>coli</i> concentrations in poultry manure and the environment.

<p>^aFor some of the samples total <i>E</i>. <i>coli</i> was not determined (*): in these cases the number of samples analyzed for ESBL-producing <i>E</i>. <i>coli</i> (a) and <i>E</i>. <i>coli</i> (b) are indicated separately (n = a; b).</p><p>^bPercentages of samples positive for the indicated bacteria.</p><p>^c Concentration ranges observed in the positive samples</p><p>^d Indicated after fly species/family (n = x, y) is the number of pools (x) and the number of flies (y)</p><p>cfu = colony forming units; Geo-mean = geometric mean; n.a. = not applicable.</p><p>ESBL-producing <i>E</i>. <i>coli</i> and <i>E</i>. <i>coli</i> concentrations in poultry manure and the environment.</p