Antimicrobial resistance (AMR) is a phenomenon that happens when bacteria become tolerant to antibiotics used to treat the infections they cause. Antibiotics are key therapeutic tools to treat many human and animal infectious diseases; consequently, keeping their activity on bacteria is fundamental for public and animal health. Aquaculture farming is known for using large amounts of antibiotics, making this activity particularly relevant in development and spread of AMR in aquatic environments. There is evidence in Chile that aquaculture activity increases the presence of resistant bacteria and their corresponding genes in marine sediments directly impacted by salmon farms; however, these evaluations are limited to few locations which hampers a generalization of these conclusions to other salmon farming sites in Chilean waters. There is also evidence suggesting that aquaculture-associated resistance genes present in marine sediments might be transferred to bacteria with pathogenic potential in humans such as Escherichia coli, but no studies have reported the presence of resistance traits in such bacteria in the marine environment from areas impacted by salmon farming in Chile. In countries like Norway, bivalve mollusks are used to monitor antibiotic resistance in Enterobacteria in the marine environment; therefore, they could also be useful for monitoring AMR associated with salmon farming and other anthropogenic sources in Chile. Sampling of bivalve mollusks represents an important logistical advantage to complex and expensive seabed sediment sampling. Los Lagos is the region with the highest historical production of farmed salmon in Chile; then, it is a suitable area to investigate the impact of aquaculture-sourced AMR in the marine environment.
The general objective of this proposal is to investigate aquaculture-associated AMR in the marine environment of Los Lagos region from an epidemiologic approach, using bivalve mollusks as sentinel organisms and E. coli as the indicator bacterium. Specific objectives include: 1) to detect, characterize and estimate the prevalence of AMR in E. coli isolated from bivalve mollusks in the Los Lagos region, through a region-level sampling; 2) to study the spatial variability of AMR in E. coli isolated from bivalve mollusks from Los Lagos region; 3) to evaluate the effect of salmon farming activity on the richness and intensity of AMR in E. coli isolated from bivalves from Los Lagos region, controlling for potential confounding factors; and 4) to determine whether significant differences exist in the accumulation of E. coli and resistant E. coli in bivalve mollusks between the sampled bivalve species, accounting for environmental and biological factors.
Bivalve mollusks samples will be obtained the 130 sampling stations set along the coast of Los Lagos by the National Program for Surveillance and Control of Harmful Algal Blooms Intoxications (red tides) coordinated by the Ministry of Health. Thirty-three additional sampling stations will be set in the same study area in order to achieve the calculated total sample size of 163 sampling stations. At each sampling station bivalve mollusks will be sampled to quantify E. coli and to detect and characterize both phenotypic and genotypic AMR in this bacterium. The study will be focused in 29 antimicrobial resistance genes (ARGs) and 20 antibiotics commonly used in salmon farming or for which resistance has been detected in previous studies. The antimicrobial susceptibility will be performed by estimating the minimal inhibitory concentrations (MICs) for each antibiotic using the VITEK2 technology. The genotypic analysis will be carried out by means of the detection target ARGs, through PCR. Spatial clustering will be examined for each antibiotic tested using MIC values and the ARG richness index; global clustering will be evaluated through the Moran’s I statistic, while local clustering will be examined by means of the spatial scan statistic. ARG richness will be modeled as a function of the local salmon farming intensity expressed as the number of active salmon farms within 10 km seaway distance from the bivalve sampling location, using a Poisson mixed-effects model in order to control for other AMR sources and important environmental variables. Similar models will be constructed using MICs as the outcome for antimicrobials that show substantial variability in this parameter. Finally, the abundance of both total and resistant E. coli (MPN/100g) found in bivalve mollusks will be modeled as a function of the bivalve species sampled, accounting for bivalve size, water temperature, local salmon farming intensity and distance to other AMR sources, using a mixed-effect linear regression model.
This study will contribute to characterize and to identify the main drivers of the environmental AMR in an area with intense salmon farming activity, and it will help to understand how this AMR can impact public health through potential pathogenic bacteria. In practice, this proposal will be the first extensive epidemiological study in this matter in Chile. Moreover, this proposal will help to determine which bivalve mollusks species are suitable to monitor AMR in environments impacted by salmon farming. All this information will be crucial to set the foundations for a future AMR monitoring program in areas of intense aquaculture.