DOI: 10.17882/75220
title: Bacterial biofilms colonizing plastics and microplastics in estuarine waters, with an emphasis on Vibrio spp. and their antibacterial resistance

description: Since plastics degrade very slowly, they remain in the environment on much longer timescales than most natural substrates and provide a novel habitat for colonization by bacterial communities. The spectrum of relationships between plastics and bacteria, however, is little understood. The first objective of this study was to examine plastics and microplastics as substrates for communities of Bacteria in estuarine surface waters. We used next-generation sequencing (NGS) of the 16S rRNA gene to characterize communities from microplastics collected in the field, and over the course of two colonization experiments, from biofilms that developed on polymers (low-density polyethylene, high-density polyethylene, polypropylene, polycarbonate, polystyrene) and glass. Field sampling and experiments were conducted in two estuarine tributaries of the lower Chesapeake Bay, the Elizabeth River and the Lafayette River, respectively. As a second objective, we concomitantly analyzed microplastics and biofilms on polymers to ascertain the presence and abundance of Vibrio spp. bacteria, then isolated three species known to be human pathogens, V. cholerae, V. parahaemolyticus, and V. vulnificus, and determined their antibiotic profiles. In both components of this study, we compared our results with analyses conducted on paired samples of estuarine water. Analyses subsequent to NGS determined fifteen different bacterial classes and 171 genera distributed across all samples. Gammaproteobacteria were the largest constituent of sequences (30%), followed by Bacteroidetes (28%) and Alphaproteobacteria (20%). Bacterial communities on polyethylene microplastics clearly differed from water samples, principally in the ratio of Alpha- and Gammaproteobacteria. In both colonization experiments, there were no differences among polymer substrates after one month with respect to the number of unique OTUs and the Shannon diversity index. Colonizing bacterial communities showed strong affinity by sampling date rather than substrate, with the exception of glass from days 2 and 4 in Colonization Experiment #2, which clustered together. Thus, there was no indication of communities specific to any polymer and those on plastics were not consistently different from those on glass. Whether from microplastics or biofilms on colonized polymer substrates, the concentration of putative Vibrio spp. was one to two orders of magnitude greater than in paired water samples. V. parahaemolyticus and V. vulnificus were cultured from every polymer type, while V. cholerae was found only on high-density polyethylene and polypropylene. Antibiotic-resistance profiles of Vibrio spp. revealed no differences between those isolated from microplastics or from biofilms on polymer substrates compared to those in paired water samples. There were, however, differences in the profiles of isolates from microplastics and those from colonization experiments, with more resistance overall in the former. This research adds to a nascent literature that suggests environmental factors govern the development of bacterial communities on a variety of polymers, more so than the characteristics of the plastic substrates themselves. In addition, this study is the first to culture V. cholerae, V. parahaemolyticus, and V. vulnificus from plastics in estuaries, reinforcing and expanding upon earlier reports of plastic pollution as a habitat for Vibrio species. The antibiotic resistance detected among the isolates, coupled with the longevity of plastics in the aqueous environment, suggests biofilms on microplastics have potential to persist and serve as focal points of potential pathogens and horizontal gene transfer.