Browsing by Author "Madhav, Hema"

Abstract Antibiotics are used to prevent microbial diseases in both animals and humans. Because of the overuse of antibiotics, the microorganism now gained the ability to resist the drugs through genetic changes. Integrons are widely known for their role in the dissemination of antibiotic resistance. The class1 integrons are mostly studied in Gram-negative bacteria of clinical strain as they are reported mostly in the human and animals. The integrons having antibiotic resistance genes are linked with mobile genetic elements which help them to disseminate by the lateral gene transfer method. Previous research has proved that the class 1 integrons have sulfonamide and tetracycline resistance gene by using Long-Range PCR, Inverse PCR, and metagenomics. However, it is not clear what other possible combination of antibiotic resistance genes the class1 Integrons may carry. My thesis focuses on the class 1 integron from wastewater (both inflow and outflow water) by Long-range PCR, which can amplify fragments more than 15kb and PacBio RS long-read sequencing. Its a novel method of combining Long-range PCR and may illuminate what other possible resistance genes the class 1 integrons carry. The antibiotics resistance genes such as CatB8, -aadA2, blaOxA-1 0, IMP-38 were amplified using our designed primers from IntI1 to QacEdelta1, thus the designed primers and the optimization of Long-Range were successful. The combination of inverse PCR and Pac-Bio sequencing was successful to amplify the antibiotic resistance genes from Class 1 integrons. The Long-Range PCR saves time and gives DNA amplified products longer than 1500kb. The purified samples from long range PCR can be studied by direct sequencing using the Pac-Bio sequencer. Thus, the future implementations of the above combination of two techniques can be very useful to study the antibiotic resistance genes in the soil and polluted water. More in-depth information about antibiotic resistance genes in class 1 integrons will help to understand their dissemination.