A total of 32 samples were collected in the central area of Spain from April 2012 to April 2014; 16 samples of manure from different broiler poultry and swine farms (poultry manure [n = 8] and pig slurry [n = 8]), and 16 samples of soil (agricultural soil periodically amended with pig slurry [n = 8] and soil from the same region not amended in the last five years or more [n = 8]). Each of the 32 samples consisted of six subsamples, pooled from each collection site. Manure samples were collected from storage tanks. Soil samples were collected at 5 - 10 cm deep. Amended soil samples were collected at least 15 days after amendment with pig slurry, the most commonly used amendment in the studied area. Each sample of nonamended soil was collected approximately 10 km apart from the rest. We compared the presence of antibiotic resistance in poultry vs. pig slurry manure and amended soil vs. NAIS soils. The sample size was determined attending to logistics reasons, selecting those broiler poultry and swine farms that allowed the sampling operations.

High Pure Template DNA (Roche Diagnostics, Mannheim, Germany) and E.Z.N.A^® soil DNA kit (Promega, Madison, WI, USA) were used to extract DNA from poultry manure and pig slurry samples, and soil samples, respectively. The presence of 16S rRNA gene and 18 selected ARGs was tested using real-time PCR (rtPCR); 18 of them based on SYBR^® Green and one based on a TaqMan^® probe (Table 1), all performed in an Eco Illumina thermocycler (Illumina, San Diego, CA, USA).

To study different profiles, we selected genes related to some of the initially most commonly used antibiotics (e.g., catA1, catA2, tet(A), tet(B), tet(C), tet(M), tet(Q), sul1, sul2, str and aadA), the currently most commonly used antibiotics in poultry and swine production (qnrB, qnrS, bla_TEM), and selected antibiotics of clinical relevance (armA, bla_CTX-M, mecA and vanA, respectively responsible to aminoglycoside-, extended-spectrum β-lactamase-, methicillin- in Staphylococcus aureus and vancomycin-resistances). It was not possible to discriminate between strA-strB genes or the different aadA alleles. Gut microbiome samples previously tested as positive for the selected genes were selected as positive controls. Unfortunately, information regarding the antibiotics used in the studied animal farms was confidential.

The 16S rRNA gene (present in all bacteria) was used to check the samples’ viability. A sample was considered validated when a 1/10 dilution of the purified DNA showed a cycle threshold less than 25. In addition, this gene was used to quantify the presence of the selected ARGs in different samples, establishing the relative concentration of the sul2 gene in comparison with the 16S rRNA gene, as follows: number of copies of sul2 gene per reaction/number of copies of 16S rDNA gene. The sul2 gene was selected to perform the relative quantification of its presence because it was one of the two ARGs detected in NAIS. Standard controls for the 16S rRNA gene and the sul2 gene were established based on previously tested samples. Purified DNA fragments were cloned into a plasmid vector (pGEM^®-T, Promega, Madison, WI, USA) and purified for a second time (Wizard^® Plus SV Minipreps DNA Purification System, Promega, Madison, WI, USA). Plasmid colonies were subcultured in LB medium supplemented with ampicillin. Total DNA was measured by a spectrophotometer. The estimated number of copies was obtained by dividing the absorbance value at 260 nm by the plasmid molecular weight (including the specific insert). Subsequently, ten-fold dilutions of quantified plasmids were evaluated by rtPCR to establish the detection limits of sul2 and 16S rRNA genes.

Chi-square test was used to compare the occurrence of ARGs between samples of poultry manure and pig slurry, and pig slurry-amended soils and NAIS. Relative

^aTaqMan probes: 5’ (6FAM); 3’ (nonfluorescent quencher). F = Forward, R = Reverse.

quantification (number of copies of ARG/number of copies of 16S rDNA) and the mean number of genes per sample among all four categories were evaluated by the Mann-Whitney U test. All statistical studies were performed with the aid of SPSS 15.0 software. Statistical significance for all tests was defined as P < 0.05.

Statistically significant differences were detected between poultry manure and pig slurry in relation to the mean number of ARGs present in the samples (P = 0.013); mecA (P = 0.012), vanA (P = 0.007), qnrB (P = 0.021), and bla_TEM (P = 0.000) genes were found more frequently in poultry manure, while tet(C) (P =

^aCalculated as follows: number of copies of ARG per reaction/number of copies of 16S rDNA gene.

0.007) and tet(M) (P = 0.021) ARGs were more frequently found in pig than in poultry slurry.

The most commonly detected ARGs in animal manure samples were tet(A) (16/16), sul1 (16/16), sul2 (16/16), aadA (16/16), tet(B) (15/16) and str (15/16), all related to some of the initially most commonly used antibiotics. Efflux pump genes―tet(A) and tet(B)―were present in almost all the animal manure samples (8/8 and 8/8 in pig slurry samples and 8/8 and 7/8 in poultry manure, respectively). The ribosomal protection protein gene tet(M) and the tetracycline efflux pump gene tet(C) were more frequently found in pig than in poultry manure, whereas tet(Q) was more frequent in both poultry manure and pig slurry samples. Based on our findings, pig slurry may be considered a major source of tet genes, followed by poultry manure. Our findings partially agree with Cheng et al. [13] , who reported tet and sul genes as the most frequently detected ARGs in animal manure.

Genes able to confer resistance to streptomycin-str and aadA- [14] and spectinomycin-aadA- [15] were detected in poultry manure and pig slurry. The presence of str genes and aadA in E. coli from poultry manure and pig slurry was previously reported [16] .

The gene catA1 was present in four out of 16 samples of animal origin (poultry manure [3/8] and pig slurry [1/8]). None of the samples presented the catA2 gene. Interestingly, the use of chloramphenicol in food-producing animals has been banned in the EU since 1994 [17] . The presence of catA1 in the absence of chloramphenicol residues to induce selective pressure has been attributed to its relationship with other plasmid-mediated ARGs [18] . Bacterial isolation was not performed in this study; therefore, we were not able to determine if different ARGs were present in the same isolate. However, we found that all cat positive samples were also positive for sul1 and sul2, which is in agreement with previously published reports [19] .

Regarding quinolone resistance genes, qnrS was present in pig slurry samples (3/8) and highly prevalent in poultry manure (6/8), while qnrB was only detected in poultry manure samples (4/8). The presence of qnr genes in poultry products is well documented, but our prevalence is considerably higher than those previously published: 3.9% and 0.9% for qnrB; 5.1% and 8.11% for qnrS, respectively [20] [21] , probably due to our methodology, more sensitive than previously culture-based studies [20] [21] . Thus, the real prevalence of qnr genes could be higher than expected.

Beta-lactamase genes bla_TEM and bla_CTX-M, were detected in poultry manure― 7/8 and 1/8, respectively―but not in pig slurry. These genes are related to extended-spectrum beta-lactamases (ESBLs), important Enterobacteriaceae resistance mechanisms against cephalosporins [22] . CTX-M-type resistance genes have replaced other families, such as TEM and SHV, emerging all over the world [23] . The presence of bla_CTX-M gene in E. coli isolated from poultry manure has been previously reported [16] . All pig slurry samples were negative, although other studies have isolated E. coli with bla_TEM and bla_CTX from pig slurry [16] .

Vancomycin resistance gene (vanA) was only detected in poultry manure (5/8). Vancomycin-resistant enterococci have been related to the use of avoparcin, which was the first growth promoter used in food-producing animals banned by the European Union, in 1997 [24] [25] . After an initial decline, the presence of vanA gene seems to persist in poultry [26] [27] , and based on our results, is still widely present in poultry manure.

The high prevalence of mecA gene, linked to methicillin resistant Staphylococcus aureus (MRSA), was noteworthy in chicken manure samples (6/8). This gene was also detected in one sample of pig slurry (1/8). To our knowledge, this is the first report of mecA gene direct detection in poultry manure and pig slurry. Colomer-Lluch et al. found the mecA gene in fecal waste samples from slaughterhouses, suggesting its transport by bacteriophages and linking it to bacteria affecting avian and swine species [28] .

There were statistically significant differences between amended and no-amended soils regarding the presence of sul1, sul2, tet(Q), and aadA (P = 0.007, P = 0.007, P = 0.007 and P = 0.021, respectively); the relative concentration of sul2 (P = 0.014), and the mean number of ARGs per sample (P = 0.009). The presence of ARGs was higher in amended soils, as well as the relative concentration of sul2, showing that these genes are probably derived from human activities, as observed by Pruden et al. [29] .

Eleven different ARGs were present in at least one manure amended soil sample: all sul and tet genes, bla_TEM, qnrS, aadA and str genes. On the other hand, only sul2 and str genes were found in NAIS (3/8 and 2/8, respectively). No soil samples presented the genes selected by their clinical relevance (armA, bla_CTX-M, mecA, and vanA). Our results differ from previous studies that detected several ARGs in both natural and anthropogenically impacted environments [29] [30] .

Gene bla_TEM (3/8) and qnrS (3/8), related to antibiotics frequently found in swine production, were detected in pig slurry amended soil, but qnrB gene was not detected in this study. The presence of bla_TEM in this type of soil was observed by Binh et al. [31] , while qnrS gene has been previously detected in other anthopogenically impacted soils, like soils irrigated with wastewater [32] .

We amplified the str gene in both amended soils and NAIS, and aadA only in amended soils. Both genes, str and aadA, have been detected in antic soil samples from the Siberian permafrost [33] , previous to the antibiotic era, so these elements could be related to other factors beside the modern use of antibiotics.

The sul2 gene was found in amended and nonamended soils, but its relative concentration was significantly higher in amended soil samples. The relative concentration of sul2 gene in agricultural soils presented the same order of magnitude as previously published [34] . It is important to remark that this gene is considered the most sensible indicator of anthropogenic/agricultural impact [29] .

Our findings demonstrate that agricultural activities may contribute significantly to the environmental spread of aadA, sul1, sul2, and tet(Q) through the use of pig slurry to amend agricultural soils. Although not significant, the presence of other tet genes, bla_TEM and qnrS seem to be higher in those soils amended with pig slurry. In addition, amended soils presented higher mean ARGs number per sample than NAIS.

As a consequence, in despite of the impossibility to determine the individual bacterial ARGs due to the lack of culture and subsequent isolation, it is possible to obtain specific patterns of anthropogenic and nonanthropogenic environments based on ARG profiles and concentrations.

Animal waste carries ARGs, some of clinical relevance (vanA, mecA, and bla_CTX-M), which may be spread to the environment through the use of animal manure to amend agricultural soils. This is the first report of direct detection of mecA in poultry manure and pig slurry. Despite the limited sample size and number of ARGs we studied, our results show the impact of anthropogenic activities on the environment through the detection of several ARGs, e.g., sul1, bla_TEM and qnrS, in manure-amended agricultural soils, while the same ARGs were below the detection limit in NAIS. Our results support the hypothesis that the detection and quantification of ARGs may serve as indicators of the “anthropogenic impact” on the environment and propose the development of specific ARG profiles of these pollutants to compare different environmental scenarios. Further studies, focusing on the consequences of anthropogenic activities on microbial populations in different scenarios are needed to elucidate the real impact of ARGs.