Cephalosprinase in a Transposition Unit Carried by Plasmid in an Environmental E. coli

Antibiotic discovery was the most significant breakthrough in medicine and allowed treatment of a wide variety of bacterial infections. However, proliferation of antibiotic resistant genes among bacteria results in many antibiotics becoming useless. The way in which bacteria develop and transfer these antibiotic resistance genes is important for understanding the origins of these genes and their prevalence, and for better understanding how to deal with them in the clinic. The hypothesis of this study was: bacteria in the environment can serve as hubs of horizontal gene transfer and can acquire and distribute antibiotic resistance genes through mobile elements such as plasmids and transposons. An environmental Escherichia coliwas isolated from Southern California surface water and designated as SW0101R. The isolate carried six plasmids (96-, 95-, 3-, 2.5-, 1.8-, and 1.4-kb). These plasmids were transformed into E. coli DH10B and selected on medium containing ampicillin. The E. coliSW0101R-AMP transformant carrying the 95 kb plasmid was obtained. The 95kb plasmid from the transformant was isolated using Qiagen kit and sent out for Illumina sequencing. The reads were analyzed using Geneious. Antibiotic disk diffusion tests were performed to observe antibiotic resistance of SW0101R and SW0101R-AMP. The tests revealed that SW0101R was resistant to ampicillin (AMP), cefotaxime  (CTX), ceftazidime (CAZ), ciprofloxacin (CIP), sulfisoxazole (SXZ) and tetracycline (TET), while SW0101R-AMP was resistant to AMP and CAZ. From plasmid sequencing, a circular contig with a size of 95,175bp (pSW0101R-AMP-95175) with G+C content of 49.9% was generated using de novo assembly. The annotation of pSW0101R-AMP-95175 using plasmid references from the National Center for Biotechnology Information database, predicted 184 protein coding sequences. The plasmid was typed as incompatibility group I1 (IncI1). The presence of genes for a type IV secretion system suggested that the plasmid was conjugative. Genes for cephalosporinase blaCMY2, an outer membrane lipoprotein, and quaternary ammonium compound-resistance protein were found adjacent to each other and linked to ISEcp1 as a transposition unit. The 4,167bp transposition unit was flanked by 7bp direct repeats (AGTAATA). A nucleotide blast was performed using the transposition unit and high similarity (99-100%) to regions from 46 different isolates was found. These isolates originated from human and farm animals from different continents. Although we initially found the transposition unit from E. coli IncI1 plasmid, it was also found in mostly IncA/C plasmids and in Salmonella enterica. These results showed the ability of this transposition unit to mobilize to different incompatibility groups of plasmids. Further analysis is necessary to better characterize its spread to environmental bacteria, which may act as a reservoir for resistance genes or other genes.