Characterization of Four Multidrug Resistance Plasmids Captured from the Sediments of an Urban Coastal Wetland

Abstract

Self-transmissible and mobilizable plasmids contribute to the emergence and spread of multidrug-resistant bacteria by enabling the horizontal transfer of acquired antibiotic resistance. The objective of this study was to capture and characterize self-transmissible and mobilizable resistance plasmids from a coastal wetland impacted by urban stormwater runoff and human wastewater during the rainy season. Four plasmids were captured, two self-transmissible and two mobilizable, using both mating and enrichment approaches. Plasmid genomes, sequenced with either Illumina or PacBio platforms, revealed representatives of incompatibility groups IncP-6, IncR, IncN3, and IncF. The plasmids ranged in size from 36 to 144 kb and encoded known resistance genes for most of the major classes of antibiotics used to treat Gram-negative infections (tetracyclines, sulfonamides, β-lactams, fluoroquinolones, aminoglycosides, and amphenicols). The mobilizable IncP-6 plasmid pLNU-11 was discovered in a strain of Citrobacter freundii enriched from the wetland sediments with tetracycline and nalidixic acid, and encodes a novel AmpC-like β-lactamase (bla_WDC-1), which shares less than 62% amino acid sequence identity with the PDC class of β-lactamases found in Pseudomonas aeruginosa. Although the IncR plasmid pTRE-1611 was captured by mating wetland bacteria with P. putida KT2440 as recipient, it was found to be mobilizable rather than self-transmissible. Two self-transmissible multidrug-resistance plasmids were also captured: the small (48 kb) IncN3 plasmid pTRE-131 was captured by mating wetland bacteria with Escherichia coli HY842 where it is seemed to be maintained at nearly 240 copies per cell, while the large (144 kb) IncF plasmid pTRE-2011, which was isolated from a cefotaxime-resistant environmental strain of E. coli ST744, exists at just a single copy per cell. Furthermore, pTRE-2011 bears the globally epidemic bla_CTX-M-55 extended-spectrum β-lactamase downstream of ISEcp1. Our results indicate that urban coastal wetlands are reservoirs of diverse self-transmissible and mobilizable plasmids of relevance to human health.

Keywords: antibiotic resistance genes; conjugation; horizontal gene transfer; mobile genetic elements; mobilizable; plasmids; self-transmissible; wetlands.

FIGURE 1

Simplified maps of the four plasmids described in this study. Color scheme: green, backbone genes (partitioning, replication, mobilization, transfer, mating pair formation and stability); gray, unidentified open reading frames; yellow, mobile genetic elements (insertion sequences, transposons, and integrons); red, accessory genes (antibiotic and heavy metal resistance). The following atypical abbreviations were used: dhps, dihydropteroate synthase; icr, isochorismatase hydrolase; pgm, phosphoglucomutase; srr, siderophore receptor.

FIGURE 2

Evolutionary relationships of WDC-1 to representatives from the other classes of β-lactamases. The phylogeny was inferred from the amino acid sequences of each protein using the Maximum Likelihood method based on the Whelan and Goldman model (Whelan and Goldman, 2001). The tree with the highest log likelihood (–9267.3106) is shown. Five-hundred bootstrap replicates were used to establish support for the tree topology and the percentage of trees in which the associated taxa clustered together is shown next to the branches for all branches with over 50% support. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Joining and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites [five categories (+G, parameter = 9.5913)]. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 32 amino acid sequences. All positions with less than 95% site coverage were eliminated. There was a total of 185 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 (Kumar et al., 2016).