بررسي حضور و سرنوشت باكتري‌هاي مقاوم به آنتي بيوتيك و برخي ژن هاي مقاومت آنتي بيوتيكي بتالاكتام به وسيله‌ي qPCR در تصفيه‌خانه فاضلاب

Investigating the presence an‎d fate of antibiotic-resistant bacteria an‎d some β-Lactam antibiotic resistance genes by qPCR in a sewage treatment plant

زيست شناسي سلولي مولكولي و ميكروبيولوژي

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

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