چكيده لاتين
Abstract
In recent decades, the extensive use of antibiotics in humans and livestock has led to the emergence and spread of antibiotic-resistant bacteria and associated resistance genes (ARGs) in the environment. Municipal wastewater treatment plants (WWTPs), as major hotspots for the accumulation and dissemination of these genes, play a dual role: while reducing the overall microbial load, they may also act as reservoirs that facilitate the horizontal transfer of resistance genes among bacteria. This study aimed to investigate the presence and variation of β-lactamase genes blaTEM and blaCTX-M in amoxicillin- and cefixime-resistant bacteria isolated from the municipal wastewater treatment plant located in southern Isfahan, Iran.
For this purpose, influent and effluent wastewater samples were collected, and resistant bacteria were isolated and identified using biochemical tests and antibiotic susceptibility assays. DNA was extracted from the resistant isolates to determine the presence of blaTEM and blaCTX-M genes through PCR analysis. In addition, to quantify the abundance of the antibiotic resistance gene blaTEM, metagenomic DNA was extracted from both influent and effluent samples, and qPCR was performed using specific primers.
Microbial enumeration revealed that the concentration of culturable bacteria in the influent was 2.78×10⁹ CFU/mL, whereas in the effluent it was 1.04×10⁵ CFU/mL, indicating an approximately log4 reduction in microbial load during the treatment process. A total of 98 isolates were obtained from the samples, among which 48 isolates (16 from the influent and 32 from the effluent) exhibited resistance to both amoxicillin and cefixime. Genetic analysis showed that among the isolates from the influent, 94% harbored the blaTEM gene and 62.5% harbored the blaCTX-M gene, whereas in the effluent these values decreased to 37.5% and 25%, respectively. The qPCR results on metagenomic DNA demonstrated that the absolute abundance of blaTEM and the reference gene 16S rRNA increased in the effluent, and the copy number ratio of blaTEM to 16S rRNA was also higher in the effluent compared to the influent. These results indicate the persistence and accumulation of this resistance gene during the treatment process.
Overall, the findings revealed that although the wastewater treatment process significantly reduced microbial density, it was not effective in completely eliminating antibiotic resistance genes. Moreover, while PCR results showed a reduction in the frequency of resistant isolates in the effluent, qPCR analysis indicated an increase in the copy number of both blaTEM and 16S rRNA genes. This discrepancy can be attributed to the methodological differences between the two assays; PCR detects only culturable bacteria, whereas qPCR quantifies the total DNA present in the microbial community, including non-culturable cells, free DNA, and mobile genetic elements. Therefore, the relative increase of blaTEM at the metagenomic level reflects the persistence and accumulation of resistant genetic material against the treatment processes
Keywords: Antibiotic, Antibiotic Resistance Genes (ARGs), Antibiotic-Resistant Bacteria, Wastewater Treatment System, β-lactam
