Salmon aquaculture and antimicrobial resistance in the marine environment

Abstract

Antimicrobials used in salmon aquaculture pass into the marine environment. This could have negative impacts on marine environmental biodiversity, and on terrestrial animal and human health as a result of selection for bacteria containing antimicrobial resistance genes. We therefore measured the numbers of culturable bacteria and antimicrobial-resistant bacteria in marine sediments in the Calbuco Archipelago, Chile, over 12-month period at a salmon aquaculture site approximately 20 m from a salmon farm and at a control site 8 km distant without observable aquaculture activities. Three antimicrobials extensively used in Chilean salmon aquaculture (oxytetracycline, oxolinic acid, and florfenicol) were studied. Although none of these antimicrobials was detected in sediments from either site, traces of flumequine, a fluoroquinolone antimicrobial also widely used in Chile, were present in sediments from both sites during this period. There were significant increases in bacterial numbers and antimicrobial-resistant fractions to oxytetracycline, oxolinic acid, and florfenicol in sediments from the aquaculture site compared to those from the control site. Interestingly, there were similar numbers of presumably plasmid-mediated resistance genes for oxytetracycline, oxolinic acid and florfenicol in unselected marine bacteria isolated from both aquaculture and control sites. These preliminary findings in one location may suggest that the current use of large amounts of antimicrobials in Chilean aquaculture has the potential to select for antimicrobial-resistant bacteria in marine sediments.

Figure 1. Locations of aquaculture and sites of sampling in the Calbuco archipelago, Chile.

Salmon farming sites are indicated by stars. The “aquaculture site” sampled in the present study (arrowhead, inset) was 20 m from the salmon farm indicated by arrow. Other sites sampled in the present study were located 0.5 km (solid triangle), 1 km (solid diamond) and 8 km (solid circle) from the aquaculture site. The latter site was off the coast of Tabón Island, an island with no aquacultural activities or other human activity, and is referred to as the “control site” in the text.

Figure 2. Culturable bacteria in sediment samples at aquaculture and control sites taken at various time points.

A. Colony forming units (cfu) g⁻¹ sediment (mean ± SE) in samples taken from September, 2008, to September, 2009, were significantly higher at the aquaculture site (closed circles) than at the control site (open circles) at all time points (P<0.001); different lower case letters indicate significant differences (P<0.05). A total of 66 samples were taken, 33 from the aquaculture site and 33 from the control site. B. Cfu g⁻¹ sediment (mean ± SE) taken in November, 2008, at the aquaculture site (0.0 km) and at sites 0.5, 1.0 and 8.0 km (control site) distant from it. Aquaculture and control sites correspond to sites shown in Fig. 2A for this date; five samples were taken from each additional site studied. Different lower case letters indicate significant differences (P<0.05, see text for details of statistical analysis).

Figure 3. Antimicrobial resistant bacteria in sediment samples from aquaculture and control sites.

Antimicrobial resistance fraction (ARF) (mean ± SE) of culturable bacteria to (A) oxytetracycline and (B) oxolinic acid in sediments from aquaculture (solid bars) and control (open bars) sites from September, 2008, to September, 2009, were significantly different between aquaculture and control sites over the entire study period (P<0.001). ARF to (C) florfenicol in sediments from aquaculture and control sites were significantly different only in November, 2008, December, 2008, and January, 2009. *, P<0.05, see text for details of statistical analysis. A total of 66 samples were taken, 33 from the aquaculture site and 33 from the control site.

Figure 4. Variation in ARF to selected antimicrobials with distance from aquaculture site.

ARF (mean ± SE) in November, 2008, to (A) oxytetracycline, (B) oxolinic acid, and (C) florfenicol in sediments at aquaculture site (0.0 km) and at sites 0.5, 1.0 and 8.0 km (control site) distant from it. ARF for aquaculture and control sites correspond to ARF shown in Fig. 3 for this date. Five samples were taken from each additional site studied. ARF to each antimicrobial were significantly greater at the aquaculture than at the control site (probabilities indicated for each antimicrobial). Different lower case letters within each panel indicate significant differences between ARF (P<0.05, see text for details of statistical analysis).

Figure 5. Antimicrobial resistance genes in unselected marine bacterial isolates and controls.

Detection of antimicrobial resistance genes in bacteria cultured from marine sediments obtained from December, 2008, to January, 2009. qnr, tet and floR genes were detected by PCR as described in Material and Methods with primers in Table 3. D15, J12, DC5, J19, DC12, D14 and D17 are bacterial isolates from sediment. −, negative control (E. coli DH5α). +, positive controls (Table 3). M, molecular weight markers.