Materials and Methods 1. Sampling settings

Samples from Tunisia comprise incoming raw sewage to WWTPs sampled in two locations between 2011 and 2014. The first location was a WWTP treating water equivalent to 20,000

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inhabitants corresponding to three towns in a pre-desert area (very warm in summer and low yearly rainfall) in South Tunisia (WWTP 1) with contribution of waste from tourist facilities.

The second location was a municipal medium-load WWTP in a metropolitan area in North Tunisia (WWTP 2), the origin of its treated influents being domestic. The third raw wastewater was collected from an abattoir slaughtering mostly sheep and to a lesser extent cattle (Fig. 1). Samples from Spain comprise incoming raw sewage to a WWTPs treating water equivalent to 500,000 inhabitants in the area of Barcelona between 2011 and 2014.

The faecal load of the samples studied as measured by the concentration of somatic coliphages (Jebri et al., 2012) was similar for samples from Tunisia and Spain showing faecal loads as reported in the Barcelona area for these types of sample (Muniesa et al., 2012). The samples were collected in sterile conditions, frozen at -80ºC and stored in dry ice until laboratory analysis. In total, 26 samples were used for total DNA extraction and 28 for DNA extraction from the viral (phage) fraction, followed by quantification by real-time qPCR.

Fig. 1. Geographic location of the three Tunisian sampling points: WWTP 1, WWTP 2 and the slaughterhouse.

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2.2. Purification of bacterial DNA

Five mL of each sample was passed through a 0.45 µm EZ-Pak^® membrane filter (Millipore, Bedford, Massachusetts), described by the manufacturer as a low protein-binding membrane.

This filter allowed the phages to pass through whilst bacteria were retained on the surface of the filter. The membrane containing retained bacteria was recovered in 5 mL of LB. The suspension was then centrifuged at 3000xg for 10 min. To recover bacterial DNA, the pellet was resuspended in 200 µL of fresh LB. DNA was then extracted using a QIAamp DNA Blood Mini Kit (Qiagen Inc., Valencia, USA), following the manufacturer’s instructions.

Bacterial DNA was resuspended in a final volume of 200 µ L and DNA concentration was evaluated using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Thermoscientifics, Wilmington, USA).

2.3. Purification of DNA in the phage particles

Recovery of phage particles was performed according to the USEPA protocol (USEPA, 2006). Briefly, 0.05 M AlCl3 was added to 100 mL of well-mixed samples to a final concentration of 0.0005 M and pH was adjusted to 3.5. After centrifugation, the pellet was resuspended in 100 mL of buffered 10% beef extract (pH 8) (LP029B; Oxoid). Polyethylene glycol (PEG) precipitation was performed by adding 50% (w/v phosphate solution pH 7.2) PEG 2000 (Sigma) to the resuspended pellet. After rigorous agitation, the mixture was kept at 4ºC overnight and then centrifuged at 8000xg for 90 min at 4ºC. The pellet obtained was then suspended in 5 mL phosphate buffer (pH 7.2) and finally filtered through 0.22 µm nitrocellulose filters. After that, extraction of DNA inside phage particles was performed as previously described (Colomer-Lluch et al., 2011a), with a few modifications, from 1 mL of virus suspension. The viral suspension was treated with chloroform (1:10) in order to avoid the presence of possible vesicles containing DNA, followed by DNase treatment (100

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units/mL) for 1 hour at 37ºC to remove any possible free DNA that might be present outside the phage particles. After that, DNase was heat inactivated at 80ºC for 10 minutes. DNA extraction was performed via proteinase K treatment and phenol:chloroform treatment (Colomer-Lluch et al., 2011a; Sambrook and Russell, 2001).

2.4. qPCR procedures

2.4.1. Standard curves

Each ARG under study was amplified by conventional PCR, purified and then cloned into a pGEM-T Easy vector for insertion of PCR products, following the manufacturer’s instructions (Promega, Barcelona, Spain), as previously described (Colomer-Lluch et al., 2011). After obtaining and purifying the plasmid construct, the DNA concentration was quantified using a NanoDrop ND-1000 spectrophotomoter (NanoDrop Technologies, Thermoscientifics, Wilmington, USA). Standard curves were generated using serial decimal dilutions of the standard carrying the targeted genes, and the CT values obtained by qPCR were plotted against log gene copy number (Colomer-Lluch et al., 20011a, b).

2.4.2. Primers and probe design

In brief, the primers and probes for each ARG (Table 1) were designed using the software tool Primer Express 3.0 and commercially synthesized (Applied Biosystems, Spain) and were used in a standardized TaqMan amplification protocol in a Step One Real Time PCR System as previously described (Colomer-Lluch et al., 2011). All probes were Minor groove binding probes with a FAM reporter (FAM: 6-carboxyfluorescein) and a non-fluorescent quencher (NFQ). All samples were run in triplicate, along with the standards, and positive and negative controls. The efficiency (E) of our reactions ranged from 95% to 100%.

161 Table 1.

qPCR primers and probes for qPCR assays used in this study Target

gene PCR Sequence Amplimer

(bp) Ref.

bla_TEM UP CACTATTCTCAGAATGACTTGGT 85 Lachmayr et

al., 2009 LP TGCATAATTCTCTTACTGTCATG

Probe 6FAM-CCAGTCACAGAAAAGCATCTTACGG-MGBNFQ

blaCTX-M-1 UP ACCAACGATATCGCGGTGAT 101

Colomer-Lluch et al., 2011a LP ACATCGCGACGGCTTTCT

Probe 6FAM – TCGTGCGCCGCTG-MGBNFQ

blaCTX-M-9 UP ACCAATGATATTGCGGTGAT 85

Colomer-Lluch et al., 2011b LP CTGCGTTCTGTTGCGGCT

Probe 6FAM – TCGTGCGCCGCTG- MGBNFQ

mecA UP CGCAACGTTCAATTTAATTTTGTTAA 92 Volkman et

al., 2004 LP TGGTCTTTCTGCATTCCTGGA

Probe 6FAM-AATGACGCTATGATCCCAATCTAACTTCCACA-TAMRA

qnrA UP AGGATTGCAGTTTCATTGAAAGC 138

Colomer-Lluch et al., 2014 LP TGAACTCTATGCCAAAGCAGTTG

Probe 6FAM-TATGCCGATCTGCGCGA-MGBNFQ

qnrS UP CGACGTGCTAACTTGCGTGA 118

Colomer-Lluch et al.,

3.1.1. qPCR controls to exclude non-encapsidated DNA

The phage particle DNA extraction protocol used was always accompanied by several controls in order to ensure that the results obtained were only due to the amplification of the DNA contained within the capsid of bacteriophage particles. Additionally to the chloroform and DNase treatments mentioned above, the following steps were performed:

3.1.2. Absence of non-packaged DNA contamination

To rule out the possibility of contamination with free DNA outside the phage particles, an aliquot of the sample taken after DNase treatment and before desencapsidation was evaluated.

At this stage, the samples were used as a template for conventional PCR of eubacterial 16S rDNA and as a template for the qPCR assay of each antibiotic resistance gene.