Nine Plasmids Discovered in a Multidrug Resistance Escherichia coli Isolate

The dissemination of antibiotic resistance genes, driven by mobile genetic elements, is a growing concern due to the increase of multiple drug resistant bacteria that cause infection. The spread of antibiotic resistance genes and their association with transposable elements have been reported, however further insight into the protein-coding content of plasmids is required. The hypothesis of this research was that the environment is a reservoir of antibiotic resistance genes carried on plasmids that are disseminated worldwide. As part of a systematic survey of E. coli from the environment we found an isolate, E. coli SW0117R, which contained 9 plasmids. The objective of this research was to sequence and annotate these plasmids. Plasmids of SW0117R were purified using Qiagen plasmid kits and sequenced by Illumina Next Generation sequencing. Plasmid de novo assembly was performed with Geneious software and annotations were carried out by comparison with sequences in the National Center for Biotechnology Information (NCBI) and Rapid Annotation using Subsystem Technology (RAST). Conjugation experiments were performed between E. coli SW0117R (donor) and E. coli J53 (recipient). The transconjugant was selected on medium containing 100 mg/L ampicillin (AMP) and 100 mg/L sodium azide (AZI). Antibiotic resistance profiles were determined by the antibiotic disk diffusion assay against 10 antibiotics: ampicillin (AMP), cefotaxime (CTX), ceftazidime (CAZ), ciprofloxacin (CIP), chloramphenicol (CHL), gentamicin (GEN), kanamycin (KAN), sulfisoxazole (SXZ), tetracycline (TET), and trimethoprim (TMP). E. coli SW0117R wild type carried 9 plasmids and was resistant to at least 7 antibiotics (AMP, CTX, CAZ, CIP, SXZ, TMP, and TET). A transconjugant had 3 plasmids (4, 31, and 95kb) and acquired resistance to AMP, CTX, and CAZ. Complete sequences of 9 plasmids from SW0117R were successfully assembled with a size range from 2.0 to 148kb. Based on coding sequences (tra, mob, and/or virB genes), 3 plasmids were found to be conjugative (31, 34, and 148kb), 3 were mobilizable (2.9, 4.0, 4.7kb), and 3 were unmobilizable (2.0, 6.2, 95kb). Three resistant-plasmids (R-plasmids) were identified: the 6.2, 95, and 148kb plasmids. The 148kb plasmid carried resistance genes to trimethoprim (dfrA17), sulfonamides (sul1), spectinomycin and streptomycin (aadA5), and quaternary ammonium compounds (qacEdelta1) arranged in a class 1 integron with a gene cluster conferring macrolide resistance (mph(A), mrx, and mphR(A)) downstream. The 6.2kb plasmid has 3 antibiotic resistance genes and 2 transposon elements. Inverted repeats of Tn5393 transposon were found in this plasmid, upstream of the aminoglycoside phosphotransferase genes, strA and strB, and sulfonamide-resistant dihydropteroate synthase, sul2. Moreover, this 6.2kb plasmid was 99% similar to other plasmids distributed worldwide. Dissemination of other seemingly unmobilizable plasmids should be further studied to understand the spread of resistance.