The Molecular Transfer Characteristics Of Multi-antibiotic Resistance Bacteria In Hospital Wastewarer And Municipal Wastewater Treatment Systems And The Correlation Analyses

Hospital wastewater and municipal wastewater treatment plant?WWTP?were widely considered as a reservoir of multi-antibiotic resistance bacteria?MARB?and antibiotic resistance genes?ARGs?,and they are important mediums that the spread of antibiotic resistance bacteria?ARB?and ARGs to the natural environment.In recent decades,with the increasing pollution of antibiotics in the aquatic environment,the diversity and abundance of ARB and ARGs were increased.At present,the systemic study of MARB and ARGs in WWTPs is lacked,especially the correlation analysis of ARGs and antibiotic resistance factor.In this study,three hospital wastewater samples and three different treatment process of municipal WWTPs were selected in two seasons?summer in January,winter in July?,and the influent,secondary effluent and dewatered sludge stages were chose as the research objects,the abundance and diversity of MARB and ARGs were performed by the standard plate culture and modern molecular biology methods,respectively.The correlation were analysed between ARGs and MARB,antibiotics,heavy metals,mobile genetic elements?MGEs?,the research results as follows:1.Detection of antibiotic and heavy metal in hospital wastewater and municipal WWTP.?1?The concentrations of tetracycline,oxytetracycline,ciprofloxacin,norfloxacin and ofloxacin were higher than other antibiotics in hospital wastewater and municipal WWTP,and the concentrations of antibiotics in hospital wastewater were higher than water samples of WWTP.The concentrations of Al,Fe and Mg were higher than other heavy metals in municipal WWTP.?2?According to the concentrations of antibiotics and heavy metals in municipal WWTP,the concentration of tetracycline,ofloxacin,oxytetracycline,norfloxacin,Cu,Al,Cr,Fe,Pb and Ti were decreased significantly from influent to effluent.However,the concentrations of metals Mn,Cd and As in effluent samples were significantly higher than the discharge standards,and the concentrations of metals Mn,Cd and As were 3.58,10.5 and 88.47 times of standard concentrations.Moreover,the concentrations of As were?significantly?higher than the standard concentrations of national regulation,and the concentrations of As were 5.89 times of standard concentrations.2.The abundance and diversity of MARB in hospital wastewater and municipal WWTPs.?1?The abundance of TCB in hospital wastewater,WWTPs influent,secondary effluent and dewatered sludge samples were 1.75×109³.31×1010 cfu/L,1.09 × 109⁵.75 × 1010 cfu/L,1.13×107³.90×107 cfu/L and 1.30 × 10⁸².97 × 109 cfu/g?dry weight,the same below?,respectively;The rates of MARB resistant three antibiotics to TCB in hospital wastewater,WWTPs influent,secondary effluent and dewatered sludge samples were 4.89%⁸.11%,0.19%².25%,0.72%⁴.82% and 0.58%⁵.26%,respectively;the rates of MARB resistant five antibiotics to TCB were 2.14%⁵.05%,0.02%⁰.47%,0.18%¹.03% and 0.25%⁰.96%,respectively;the rates of MARB resistant eight antibiotics to TCB were 0.12%⁰.42%,0.0036%⁰.2698%,0.0345%⁰.5680% and 0.0851%⁰.6140%,respectively.The numbers of TCB and MARB between hospital wastewater and municipal WWTPs influent were at same order of magnitude.The rates of MARB to TCB in hospital wastewater were higher than WWTPs.?2?Through the comparison analysis,it was found that the abundance of TCB and MARB were significantly reduced 1 4 logs from the influent to the secondary effluent.By comparing the wastewater samples of two seasons,the rates of MARB in July samples?extreme?significantly higher than these of January samples.The rates of MARB in secondary effluent samples were significantly higher than these of influent samples.?3?The bacteria in hospital wastewater were concentrated at Arcobacter,Acinetobacter,Bacteroides,Paludibacter,Macellibacteroides,Aeromonas,Sulfurospirillum,Faecalibacterium,Pseudomonas and Prevotella₉,and culturable MARB species are mainly classified in Escherichia,Acinetobacter,Stenotrophomonas,Myroides,Enterococcus,Stenotrophomonas,Alkaligenes and Brevundimonas.It was showed that MARB species are mainly classified in Escherichia,Acinetobacter,Stenotrophomonas,Pseudomonas,Brevundimonas,Myroides,Proteus,Morganella,Arthrobacter and Arcobacter.Most of MARB speices were G-bacteria,and belong to pathogenic bacteria or opportunistic pathogen bacteria.3.The abundance and diversity of MGEs and ARGs in hospital wastewater and municipal WWTPs.?1?According to the high-throughtput qPCR results,the detection rates of ARGs in hospital wastewater and municipal WWTPs were 73.60% 89.89%,Among them,the ?-lactam,tetracycline and aminoglycoside resistance genes were highly detected.?2?The absolute abundance of ARGs were quantified by qPCR method in July samples: the gene copies of 16 S rRNA in hospital wastewater,WWTPs influent,secondary effluent and dewatered sludge samples were 0.88×10⁸².26×10⁸ copies/ml,1.22 × 10⁸³.02 × 10⁸ copies/ml,1.56 × 107⁵.01 × 107 copies/ml and 1.08 × 1010⁴.36 × 1010 copies/g?dry weight?,respectively;the gene copies of MGEs were 0.76×10⁸⁴.8×10⁸ copies/ml,3.08 × 106⁶.22 × 107 copies/ml,1.78 × 106².01 × 107 copies/ml and 1.46 × 106¹.40 × 109 copies/g,respectively;the gene copies of tetracycline resistance genes were 5.20×106⁸.80×107 copies/ml,1.15 × 106⁶.44 × 106 copies/ml,1.87 × 104³.78 × 105 copies/ml and 5.76 × 103⁸.72 × 103 copies/g,respectively;the gene copies of macrolide resistance genes were 1.18×106³.30×106 copies/ml,1.27 × 106¹.17 × 107 copies/ml,3.63 × 104³.73 × 105 copies/ml and 1.97 × 105¹.73 × 10⁸ copies/g,respectively;the gene copies of sulfonamide resistance genes were 1.43×107⁷.76×107 copies/ml,9.28 × 106¹.22 × 107 copies/ml,4.31 × 105¹.61 × 106 copies/ml and 6.13 × 10⁸².02 × 109 copies/g,respectively;the gene copies of quinolone resistance genes were 3.14×104⁶.90×104 copies/ml,6.45 × 104¹.35 × 105 copies/ml,4.29 × 103⁸.02 × 104 copies/ml and 2.16 × 104³.65 × 105 copies/g,respectively.?3?Comparison with influent samples,the absolute abundance of MGEs,tetracycline,macrolide,sulfonamide and quinolone resistance genes were decreased 0¹.27 logs,0.48².54 logs,0.53².51 logs,0.76¹.45 logs and 0¹.27 logs.The relative abundance of MGEs was up to 0.75,this showed that at least three-quarters of bacterial gene contains MGEs.?4?The relative abundance of MGEs,tetracyclines,macrolides,sulfonamides and quinolones resistance genes in hospital wastewater were 0.86².12,0.05⁰.39 and 0.13⁰.23,which were higher than WWTPs samples.Through comparative analysis,the orders of relative abundance were MGEs > sulfonamides > tetracyclines > macrolides > quinolones.4.Correlation analysis between MGEs,ARGs and MARB,environmental pullution factors in WWTPs.According to correlation analysis results,the abundance of MARB and many ARGs have significant positive correlation.The abundance of intI3,sul1 and sul2 were?extreme?significantly correlated with the abundance of trimethoprim,tetracycline,ofloxacin,oxytetracycline,norfloxacin,sulfamethoxazole,cefalexin,ciprofloxacin,spiramycin and erythromycin.The abundance of many ARGs have significantly positive correlation with the concentration of metals.