Characterization of plasmids associated with antibiotic resistant bacteria in the North-West Province
Antibiotic release in natural environments select for antibiotic resistant bacteria. These antibiotic resistant bacteria are in many cases associated with plasmids, containing one or more resistant genes. Plasmids are mobile genetic elements and are involved in transmitting the antibiotic resistance from bacterial species off-spring or to other bacterial species. These plasmids may also transfer virulence genes from one bacterial strain to another, rendering non-pathogenic strains with virulence capabilities. This is a significant public health concern. The present study aimed to characterize plasmids associated with antibiotic resistant bacteria in selected surface water systems in the North West province, South Africa. Several multiple antibiotic resistant bacteria previously isolated from these water sources were screened for the presence of plasmids. A total of 20 parental plasmids were successfully isolated from 20 multiple-antibiotic resistant (MAR) bacterial species using the traditional alkaline lysis method. Plasmid DNA were transformed into 10-β E. coli host strain. Transformants was selected using one of the antibiotics in the resistance profile of the original MAR parental strain for selection. Furthermore, antibiotic resistance profiles of the transformants were determined and compared to those of parental strains. Finally, polymerase chain reaction (PCR) amplification and sequencing analyses were used to determine whether genes responsible for antibiotic resistance could be amplified. In addition, PCR was also used to amplify the incompatibility (Inc) group markers and classify these accordingly. Selected bacterial strains were resistant to two or more antibiotics of different classes. Among all the isolates, resistance patterns were in this specific order ampicillin (17/20), tetracycline (12/20), erythromycin (11/20), kanamycin (9/20), streptomycin (5/20), neomycin (4/20), and the least resistance to chloramphenicol (1/20). Results indicate that the Schoonspruit river had a generally diverse resistance to various antibiotic groups, compared to the Harts river and Barbers Pan. All selected plasmids belonged to the IncP group. It is known that this group of plasmids are responsible for conferring resistance to a broad spectrum of antibiotics due to the accessory modules or antibiotic resistance genes they may contain. Amplification of antibiotic resistance genes, only detected those encoding β-lactamases (ampC) and efflux pumps (tetA), but no genes encoding aminoglycoside resistance (e.g. nptII genes). Furthermore, susceptibility profiles of parental strains differed from the transformed E. coli plasmids. Suggesting that the specific phenotype was not entirely encoded by genetic elements on the plasmid. Other mechanisms may thus be responsible for the resistance phenotype. In this work, the IncP plasmid harbouring these antibiotic resistance genes were mainly isolated from Enterobacteriaceae family. Plasmids harbouring the ampC genes were the only ones that were able to transform through electroporation with sufficient transformation efficiencies that ranged between 3.1 X 108 and 7.1 X 108 transformants per microgram DNA. Both pHR2 and PSR8 were somewhat smaller than pHR5 and had higher transformation efficiencies. However, further optimization is advised using a wider range of bacterial host strains as this may also influence the uptake of plasmid DNA. Furthermore, this study also demonstrated that these plasmids can be transferred among bacteria through the bacterial transformation process. Therefore, plasmids belonging to the IncP group may be responsible for the rapid dispersal of these antibiotic genes in the aquatic environment in the North West province. This may possibly be a hazard for human and animal health as these types of plasmids may confer resistance to broad spectrum antibiotics.