Supplementary Information: Antibiotic resistance in Vibrio-like bacteria is common on Cape Cod, MA beaches

Abstract

Antibiotic resistance (AR) is a natural process, enhanced by anthropogenic antibiotic use. Natural environments, like the ocean, act as reservoirs of resistance; but until recently, little research has examined their dynamics. Six beaches on Cape Cod, MA, with varying human impacts, were sampled over one year on nine occasions. Vibrio-like bacteria were isolated from wet sand, dry sand, and water from each beach and tested for sensitivity to five antibiotics (amoxicillin/clavulanic acid, ciprofloxacin, doxycycline, oxytetracycline, and trimethoprim) using the disk diffusion method. 73% of isolates showed resistance to at least one antibiotic, and resistance was persistent over time, space, and sample type. Isolates commonly exhibited trimethoprim, ciprofloxacin, and/or amoxicillin resistance. 16S ribosomal DNA amplicon-based community structure varied along with the dominant operational taxonomic unit (OTU). Permutational multivariate analysis of variance (PERMANOVA) indicate that resistance patterns, prevalence, and bacterial community composition were often related to month of sampling. Seasonal environmental variables also explain AR and community structure data. Distance based linear models (DistLM) using arcGIS land use variables reflecting homogeneity in land use. Estimates of Vibrio-like resistant bacteria range from 57 to 980 cells per ml water, accounting for 0.00057-0.0098% of the total bacteria encountered with beach water contact. These results illustrate that resistance to antibiotics by Vibrio- like bacteria is widespread on local recreational marine beaches. Although these resistant bacteria are a small percentage of the total bacteria, they may represent a potential public health issue through the introduction of resistance genes into human microbiomes during recreation or shellfish consumption.