Antibiotic resistance in Aeromonas hydrophila associated with exposure to low levels of oxytetracycline

Antibiotic resistance (AMR) in aquatic bacteria affecting aquaculture has been a growing concern given the potential for mixing of bacterial populations in the aquatic environment and exposure to different pharmaceuticals from drugs used in aquaculture, as well as wastewater effluent and agricultural run-off. To better understand the mechanism for AMR in a common aquatic fish pathogen exposed to low dose antibiotics we monitored the genetic changes, as well as gene expression, in Aeromonas hydrophila as the bacteria was exposed to incremental doses of oxytetracycline (OTC), a commonly used drug in aquaculture. We were able to render all three isolates of our original A. hydrophila resistant to therapeutic levels of OTC (i.e. =100ppm). The relatively quick phenotypic adaptation (often less than 3 days) to different OTC concentrations was very similar across our replicates. Our whole genome sequencing data and transcriptome results suggested several genes underwent point mutations across all replicates. Further differential gene expression was observed and likely impacted several pathways which may explain the progressive resistance to OTC associated with incremental exposure to the drug. The specific mutations consistently identified in isolates exposed to OTC were on AHA_ 2785 (associated with an outer membrane protein), AHA_2910 (involved in the efflux pump mechanism), and AHA_0308 (associated with the small ribosomal subunit protein S10). The pathways involved in the differential gene expression included efflux- pump mechanisms, outer membrane proteins, and ribosomal protein OTC target. Our findings support the notion that AMR can occur via genetic regulation of several intrinsic mechanisms within a bacterial population. This finding could have implications in aquaculture where bacteria such as A. hydrophila can be exposed to varying levels of antibiotics during in-feed treatments. Overall design: We trained original Aeromonas hydrophila ATCC9766 to get resistance to different levels of oxytetracycline (i.e., 5ppm, 20ppm, 50ppm, 100ppm, and 200ppm). The data contains all RNAseq samples from original Aeromonas hydrophila ATCC9766 (R0), and all the resistant Aeromonas hydrophila (i.e., R5, R20, R50, R100, and R200).