Environmental E. Coli Isolates Carrying IncI1 Plasmids Transfer Resistance to Antibiotics

Escherichia coli are a common problem in the clinical setting due to their ability to cause urinary tract infections, bloodstream infections, and gastroenteritis. These bacteria become harder to treat by attaining multiple antibiotic resistance genes like beta-lactamase genes from other bacteria through horizontal gene transfer of plasmids that are grouped into incompatibility types IncFII, IncI1, and IncA/C. These resistance plasmids studied from clinical samples usually contain more than one antibiotic resistance gene, but the study of their resistome from the environment is minimal. The objective of this study was to characterize IncI1 plasmids with genes from environmental isolates. Environmental E. coli isolates’ (n=22) antibiotic resistance profiles were tested using disk diffusion assays against 10 antibiotics: ampicillin (AMP), kanamycin (KAN), gentamicin (GEN), cefotaxime (CTX), ceftazidime (CAZ), ciprofloxacin (CIP), chloramphenicol (CHL), sulfisoxazole (SXZ), trimethoprim (TMP), and tetracycline (TET) and the resistance profile  was determined by following the Clinical Laboratory Standards Institute (CLSI) guideline. Plasmids were isolated and transformed from competent E. coli DH10B cells. Transformants were grown on selective Tryptic Soy Agar (TSA) supplemented with AMP, GEN, or TET. Conjugation experiments were performed using sodium azide resistant E. coli J53 as a recipient. The plasmids of wildtypes, transformants, and transconjugants were separated by gel electrophoresis for 5 hours, stained with ethidium bromide, and visualized under ultraviolet light. 19 of 22 wildtypes were resistant to more than 3 antibiotics making them multidrug resistant and the most common resistances were to AMP (95.5%), SXZ (72.7%), and TET (77.3%). One wildtype was resistant to all tested antibiotics. These E. coli wildtypes were shown to possess multiple plasmids with range size from 1 to 220 kb including an IncI1 plasmid. The size of IncI1 plasmids were between 86 to 210 kb carrying at least one antibiotic resistance gene as suggested from antibiogram of transformant. All transformants (n=22) only possessed a single IncI1 plasmid and were resistant to AMP (66.2%), TET (40.9%), SXZ (31.8%), GEN (22.7%), CTX (31.8%), CAZ (32%), KAN (4.6%), and TMP (4.5%). Additionally, 55% of transformants were resistant to more than 3 antibiotics. The conjugation experiment showed that IncI1 plasmid was transferable to a donor E. coli J53. Frequency of transconjugation was more than 10^-6 per recipient and donor. Plasmid analysis of transconjugants showed that they carried not only an IncI1 plasmid, but also other unanalyzed plasmids as well. This study suggests a possible reservoir for antibiotic resistance genes on IncI1 plasmids that could be spread to pathogenic E.coli or other Enterobacteriacea. Analysis of IncI1 plasmids from other Enterobacteriaceae will be essential for further studies.