The widespread antibiotic resistance genes (ARGs) severely jeopardizing human health have been widely concerned. Wastewater treatment plants (WWTPs) are commonly considered the important sources and sinks of ARGs due to their roles in depositing ARGs and provoking ARGs' proliferation. Current WWTPs often use biological treatment to remove pollutants, whereas the purification efficacy of ARGs is unclear. Thus, this study investigated the seasonal changes of ARGs (tetA, tetC, tetM, tetO, tetQ, tetW, and tetX, as representative) and a class of integron (intI1) in the sludge of different treatment units of four WWTPs to understand the role of WWPTs in ARGs spread. It is found that ARGs abundance varied between summer and winter depending on the types of ARGs and treatment units. The cumulative abundance of ARGs in the same treatment unit showed no obvious difference between seasons. By comparison, ARGs abundance was generally lower in the anoxic unit than in the oxic unit. Moreover, the WWTPs with Cyclic Activated Sludge System (CASS) process reduced the amount of ARGs in sludge, while the WWTPs with Anaerobic-Anoxic-Oxic (A²O) increased ARGs abundance. In conclusion, this study demonstrated that the excess sludge still posed a risk for ARG spread after biological treatment.

Abstract: Honey is a natural product that has been widely used for its therapeutic effects. Honey is effective against pathogenic bacteria in inhibiting planktonic antibiotic sensitive strains and antimicrobial resistant organisms. To date, there is no comparative study of the antibacterial activity of Citrus honey (CH) and Jabali honey (JH) with that of Manuka honey (MH). Therefore, the purpose of this study was to conduct such study and to compare the antibacterial activity of CH and JH with that of MH against Pseudomonas aeruginosa. The antibacterial, antibiofilm and antivirulence activities of CH, JH and MH against P. aeruginosa were investigated by agar well diffusion, Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), time-kill curve, disruption of microcolony, microtiter plate and reverse transcription-quantitative Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR). Agar inhibition assay showed that CH, JH and MH at 20% has antibacterial activity against P. aeruginosa with an inhibition zone of 14.1±0.1 mm, 12.2±0.2 mm and 10.9±0.1 mm respectively. The results showed that the MICs value of CH and JH was 25% compared to MH (12.5%) and the MBCs value of CH and JH was 50% compared to MH (25%) against P. aeruginosa. In addition, the MIC₅₀ and MIC₉₀ for CH and JH were 25% and 50% respectively compared to MH (MIC₅₀; 12.5% and MIC₉₀; 25%) against P. aeruginosa. All tested honeys were found to disrupt the microcolony formation in P. aeruginosa. In time-kill curve, treatment of P. aeruginosa with 2×MIC (Minimum inhibitory concentration) of MH, CH and JH for 12-hours resulted in reduction in colony-forming unit (CFU/ml). The lowest concentration of 20% of MH, CH and JH was found to inhibit and eradicate biofilm formation. RT-qPCR analysis revealed that the expression of all genes (oprB, oprC, fleN, fleQ, fleR, lasR and lasI) in P. aeruginosa were downregulated after exposure to all tested honeys. Among the all-tested honeys, MH showed the highest total antibacterial, antibiofilm and antivirulence activities. This study indicates that CH and JH has antibacterial and antibiofilm activities compared with MH due to a decrease in expression of essential genes associated with P. aeruginosa.