چكيده لاتين
The widespread use of antibiotics in human societies and the subsequent entry of these compounds and Antibiotic Resistant Bacteria (ARBs) into the environment, particularly Wastewater Treatment Plants (WWTPs), has become a significant environmental and health challenge. This research aims to identify and analyze bacteria and antibiotic resistance genes (ARGs) in the Shahin Shahr wastewater treatment plant with the goal of evaluating the risk of transmission. In this research, sampling was performed at three points in the Shahinshahr municipal WWTP: influent, effluent, and activated sludge. To isolate resistant bacteria, samples were cultured on R2A agar containing the antibiotics tetracycline, ciprofloxacin, erythromycin, and amoxicillin. Colonies were counted after incubation at 27∘C and 37∘C. Bacterial identification was done by examining morphological features, classical biochemical tests, and API strips (for Enterobacteriaceae). Antibiotic susceptibility was also assessed using the disk diffusion method based on the CLSI standard. In the molecular section, DNA extraction was performed using the boiling method, purity and quantity were assessed with Nanodrop, specific primers for the ermB and qnrS genes were designed, PCR was performed, products were checked by agarose gel electrophoresis, and final confirmation was done by Sanger sequencing and analysis in the NCBI database. The results showed that Shahinshahr municipal wastewater hosts diverse and active bacterial communities with the potential for multidrug resistance to antibiotics. The results showed that the highest resistant colony counts were observed in the activated sludge (approximately 54%), followed by the influent sample (approximately 45%), and the lowest amount was found in the effluent of the treatment plant (approximately 0.001%). Morphological and phenotypic results also suggested a predominance of Gram-negative bacteria, especially Escherichia coli, and a significant presence of Gram-positive bacilli and coccobacilli. DNA was extracted from most isolates with appropriate concentration and purity, and specific primers for the ermB and qnrS genes were designed and validated. PCR results indicated that these genes were present in the majority of Gram-positive and Gram-negative isolates, and molecular investigations confirmed that the phenotypic resistance to erythromycin and ciprofloxacin was consistent with the widespread presence of the ermB and qnrS genes. Specifically, isolates C3 (Escherichia vulneris, Gram-negative) and A21 (Bacillus sp., Gram-positive) were selected for sequencing. BLASTn results showed that the obtained sequences had a high match with reference sequences, and the qnrS and ermB genes in isolate C3 and the ermB gene in isolate A21 were confirmed by Sanger sequencing, verifying the accuracy of the reactions and the specificity of the primers. Overall, Shahinshahr municipal wastewater is a dynamic environment rich in resistant bacteria, where the persistence and dissemination of antibiotic resistance are likely, and continuous monitoring along with the development of appropriate management strategies is essential to reduce the spread of this resistance.
