All protocols of experimental design were approved by the ethics and biosecurity committee of the company and the Animals Ethics Committee of Universidad San Francisco de Quito before the study.
Animals
A completely random, balanced fixed-effects double-blind study was conducted in two generations of pigs housed in a farm in Ecuador. The number of animals was calculated with a statistic power test of 87% considering the probable loss of experimental units (replacement sows). The study was conducted under strict biosecurity conditions. Twenty 70-day female PCI 1050 pigs were randomly assigned to each of the 2 groups. Sows were raised in a Site 1 farm where they were inseminated and carried out their pregnancies. One group containing 10 female pigs (the control group) was fed with conventional feed formulation with antimicrobial additives (group A) and, and the other comprised 10 female pigs which were fed without antimicrobial additives in the feed (group B).
Treatments were maintained during the whole study period (S1, supplementary materials). Three sows from treatment A and 4 sows from treatment B farrowed at the same time (there were no differences in litter numbers between treatments) (S2. Supplementary materials), piglets were placed with their corresponding mothers. When piglets reached 20 days, they were weaned and housed in another facility where they were distributed into two separate pens for group A (n = 40) and B (n = 40) and continued with treatments of their respective mothers. Each piglet was identified with a numbered earring. Once piglets were 70 days, they were moved to site 3 for fattening (71d to 170 d) (S2, supplementary materials); animals were kept under the same treatment throughout this time. The productive performance was observed based on dairy weight gain along the study period and after slaughtering the carcasses weight was compared. Vaccines were administered to all animals and the antimicrobial treatment was administered under veterinarian supervision only to animals that have any diagnosed infection. Antimicrobial additives used are described in Table 1.
During the weaning and fattening phases, each pen grouped 32 piglets. Pig density was 0,45 m2/pig in the weaning phase and 0,90–1,0m2/pig at the fattening phase. Animals from each group were monitored for 150 days (Figure 1). The type and antimicrobial concentrations in feed changed over time and have been used routinely in the farm for the two previous years (Table 1). Animals were released from the experiment at day 150 and they were slaughtered (electric stunning) at day 170.
Housing and husbandry
The maternity and weaning phases (piglets until 70 days) are carried out in pens with plastic slatted flooring, the pens for fattening phase were located in a different facility and had cement floor which was washed daily. In all growing phases, pens were protected from the sun with roof, ceiling was at 2,5m high. Number of cages are described in Fig, 1.
Tap water and feed were administered at libitum, temperature ranges from 20 to 26°C. All experiments were performed under veterinary supervision. If any sick animal was detected it was immediately spared in a different cage to be treated and eliminated from the study.
Samples and phenotypic analysis
Two Rectal swabs were taken from each sow and a randomly selected piglet from each pen during one productive cycle (Figure 1). Swabs were maintained on ice for transportation to the lab facilities within 2h after collection. Intestinal coliforms were used as a microbial indicator of phenotypic resistance.
For quantification of coliform resistant bacteria in intestinal microbiota of pigs, MKL medium was supplemented with 4 mg / liter of trimethoprim and 76 mg /liter of sulfamethoxazole (SXT) [50], or 32 mg/liter of tetracycline [51], or 16mg/liter of ampicillin according to protocols described previously [52].
Swabs were eluted in 5mL of sterile tryptic soy broth (TSB) diluted in phosphate-buffered saline solution (PO) (0.0169M KH2PO4, 0.0719M K2HPO4, pH 7,2) [51]. A 0.1mL aliquot of this solution was serially diluted in 0.9 mL of PO until 10–3 and 0.1mL of each dilution was plated onto the surface of MKL plates with and without antimicrobials [52] ( S3. Supplemented materials). We estimated the ratio of resistant coliforms by counting the number of colonies in MKL plates with antimicrobials divided by the colonies in MKL plate without antimicrobials [29]. Only plates with coliform counts greater than 4 colony forming units (CFU) were recorded (S3, supplementary materials).
Antimicrobial susceptibility test
One lactose fermenting (coliform) colony from each plate was isolated and stored at –80°C in TSB with 30% glycerol [34]. Antimicrobial susceptibility tests were performed using AMP ampicillin (10g), TET tetracycline (30g), SXT trimethoprim-sulfamethoxazole (1.25/23.75g), GEN gentamycin (10g), AMC amoxicillin-clavulanic ac. (20/10g), CIP ciprofloxacin (5g), CHLOR chloramphenicol (30g) and COX ceftriaxone (30g) as representatives of the most used families of antibacterial drugs used in health care [53, 54]. The Kirby Bauer test was carried out following CLSI (Clinical & Laboratory Standards Institute) guidelines.
Molecular analysis
For molecular analysis, swabs were frozen at –80°C until they were used. DNA from swabs taken for each pig was isolated using MO BIO Power Soil DNA Isolation Kit (MO BIO, 12888–100) using swab dilution material in bead solution buffer and following manufacturer instructions. Quality and quantity were evaluated using nanodrop (Thermo Scientific) and Qubit dsDNA HS (Thermo Fisher Scientific, Oregon, USA). From sows’ samples, mcr-1 PCR amplification was performed used the conditions described previously [18]. One fecal pooled sample, from each sampling point (6 from A and 6 from B group) was analyzed in duplicate with high throughput qPCR. WaferGen SmartChip Real-time PCR system was used to detect 384 genes, 338 are informative for AR genes or MGE. Primers for these genes and associated HT- qPCR assay were designed, used, and validated in the previous studies [35, 55–57], and primer set was update recently [58] (Suppl. 1). The genetic richness was defined as the number of AR genes found in a niche.
Statistical analysis
All collected data were registered in MS EXCEL software and descriptive and inferential statistical analysis were performed in INFOSTAT (Statistic Software Vs 2017). The impact of the antimicrobial restriction on coliforms count, on the susceptibility patterns of isolates, and animal performance was compared with T-test and Chi-Square respectively (≤0.05). HT-qPCR data were analyzed according to previously established methods [35, 59]. Specifically, ∆∆CT method was used to normalize and calculate the fold change. Moreover, the relative abundance of ARGs was calculated with normalization to the universal 16S rRNA; estimated from the Ct value with a conservative threshold Ct of 30 as the gene copy detection limit. Calculated data represent the copy number per 16S rRNA gene copy. QIUcore Omics Explore 3.4 software was used to show heat maps.