Evalu luation of f Antib ibiotic ic Resistance in in a - PowerPoint PPT Presentation

Evalu luation of f Antib ibiotic ic Resistance in in a Pharmaceutical Wastewater of China and a Novel Nitritation-Anommox Process for Autotrophic Nitrogen Removal Jiane Zuo, Ph D, Professor School of Environment, Tsinghua University Oct. 12, 2015

 Introduction of Tsinghua University;  Antibiotic Resistance in Wastewater;  Novel Nitritation-Anammox Process  Summary

Location of Tsinghua University Tsinghua University Peking University

Campus of Tsinghua University Total area: 392.4 ha

Beautiful Campus Spring Water Campus River Winter Campanile Summer Blossoms Autumn Leaves Corridors in the President Office

Headquarter Office The Old Gate Tsinghua School Auditorium

School of Environment School of Law School of Life Science School of Medicine

The University Library Students Sports Ground Indoor Swimming Pool Student Dormitories

Statistics of Tsinghua University (2014) Number Students in total 45,237 Undergraduates 15,692 (1420*) Master students 18,296(951*) Ph.D students 11,249(291*) *: International students

Statistics of Tsinghua University (2014) Number Faculty and Staff 5,985 Members of Chinese Academy of 43 Science Members of Chinese Academy of 33 Engineering Full Professors 1,442 Associate Professors 2,138

 Introduction of Tsinghua University;  Antibiotic Resistance in Wastewater;  Novel Nitritation-Anammox Process  Summary

Background  Problem of antibiotic resistant bacteria (ARB) and resistance genes (ARG) has received increasing concern ARB and ARG in the environment  Low concentrations of antibiotics in the environment can induce resistance to bacteria in long term Direct way Indirect way by by drinking, food chain swimming, transmission etc.  ARB and ARG have been detected in different environments Human acquired antibiotic resistance – Increasing difficulty of curing disease

Background  Pathways of antibiotics entering the environments Antibiotic Municipal Sewage Hospitals producing wastewater Medical wastewater Municipal wastewater Antibiotic producing treatment plant treatment plant wastewater treatment plant Discharge of Effluents Landfill Agricultrual Surface Water Activity Soil Ground Water

Background  Pathways of antibiotics entering the environment Antibiotic Municipal Sewage Hospitals producing wastewater Medical wastewater Municipal wastewater Antibiotic producing treatment plant treatment plant wastewater treatment plant An important source of antibiotics: Discharge of Effluents Wastewater Discharge Landfill Agricultual Surface Water Activity Soil Underground Water

Background  Focusing on antibiotic producing wastewater in China  Antibiotic producing wastewater contains very high concentrations of antibiotics  China is now one of the world’s largest antibiotic manufacturing countries, discharging 540 million tons of pharmaceutical producing wastewater in 2013. - 《 China Statistical Yearbook on Environment -2014 》

Background Objectives - Obtain a general understanding to the levels of antibiotic resistance in municipal wastewater, cephalosporin producing wastewater and receiving river - Investigate the effect of cephalosporin producing wastewater discharge on receiving river - Explore the influence of wastewater treatment on antibiotic resistance

Material and Method  Sampling sites The cephalosporin producing wastewater treatment plant and municipal wastewater  treatment plant were located in A city, China - 20 km apart. Both of the two wastewater treatment plants use the two-stage biological oxidation  processes Wastewater were sampled from the equalization tanks and effluents of the final  sedimentation tanks of the two wastewater treatment plants, respectively Cephalosporin producing Municipal wastewater wastewater treatment plant treatment plant

Background River sediment samples that receives cephalosporin wastewater effluent were taken  at the upstream 200 m and downstream 2000 m from the outfall, respectively Wastewater samples were labeled as:  Municipal wastewater influent (MWI) • Municipal wastewater effluent (MWE) • Raw cephalosporin producing wastewater (RCPW • Treated cephalosporin producing wastewater (TCPW) • Upstream river sediment (URS) • Downstream river sediment (DRS) •

Material and Method  Antibiotic susceptibility test - Etest  Step 1. Isolation and Identification of Enterobacter • Enterobacter was chosen as the indicator microorganism • pure colonies were identified by the API 20E system (bioMerieux, France)

Material and Method  Antibiotic susceptibility test - Etest Step 1. Isolation and Identification of Enterobacter  Enterobacter was chosen as the indicator microorganism • pure colonies were identified by the API 20E system (bioMerieux, France) • Step 2. Antibiotic susceptibility tests of isolates using E-test strips (bioMerieux, France)  Each Enterobacter was tested its minimum inhibition concentration (MIC) to seven antibiotics • Sulfonamides β -lactam class Aminoglycosides Quinolones potentiator Semi-synthetic Cephalosporins penicillins Gentamicin Levofloxacin Cefuroxime Ceftriaxone Trimetroprim Ampicillin Amoxicillin (GTM) (LVX) (CTR) (TMP) (AMP) (AMC) (CXM) 阿莫西林 头孢呋辛 头孢曲松 左氧氟沙星 左氧苄氨嘧啶 氨苄西林 庆 大霉素

Material and Method

Material and Method  Quantification of bla TEM-2 gene - SYBR Green I real-time qPCR Step 1. Distraciton of total DNA  using MoBio PowerWater DNA Isolation Kit • Step 2. qPCR (7900, Applied Biosystems, U.S.)  blaTEM-2 (Gene bank No. HQ162131.1) • Primer sequence: • bla TEM-2 - F: 5’ -AAGCCATACCAAACGACG- 3’ bla TEM-2 - R: 5’ -TTTATCCGCCTCCATCCA- 3’ 16SrRNA- F: 5’ -CCTTGAGGTAGTTGGGTA- 3’ 16SrRNA- R: 5’ -CGTTTGGAGATTAGCG- 3’ The R 2 value of the 6-point standard curve (10-fold serial dilutions, 109 to 104 copy • numbers) for qPCR was 0.995 The amplification efficiency of bla T EM-2 was 96.9% •

Material and Method  Quantification of bla TEM-2 gene - SYBR Green I real-time qPCR Step 1. Distraciton of total DNA  using MoBio PowerWater DNA Isolation Kit • Step 2. qPCR (7900, Applied Biosystems, U.S.)  Reaction system Reaction s ystem Reaction procedure Reaction p rocedure • 95  C, 2 min 2 × SYBR • 10 µL of qPCR Mastermix (Roche, Switzerland) • 40 cycles of 15s at 95  C, 1 min at 60  C • 0.4 µL of each primer (10 µM) • extension at 60 ° C and a fluorescence acquisition step at 60 ° C • 1 µL of template DNA • 8.2 µL of ddH 2 O • final melting followed as: 15 s at 95 ° C, 1 min at 60 ° C and 15 s at 95 ° C.

Material and Method  Qu Quan anti tificati fication on o of f bla bla TEM EM-2 gene gene - SYBR SYBR Green een I real eal-ti time me q qPCR PCR Step 1. Distraciton of total DNA  using MoBio PowerWater DNA Isolation Kit • Step 2. qPCR  The R 2 value of the 6-point standard curve (10-fold serial dilutions, 109 to 104 copy • numbers) for qPCR was 0.995 The amplification efficiency of bla T EM-2 was 96.9% •

Result and Discussion  Antibiotic resistance levels of Enterobacteriaceae Percentage of antibiotic-resistant Number of total Number of Sampling Enterobacteriacea (%) heterotrophic bacteria Enterobacteriacea site (CFU/mL) isolates GTM LVX TMP AMP AMC CXM CTR 4.9 × 10 8 RCPW 79 13.9 10.1 16.5 57.0 39.2 67.1 57.0 2.7 × 10 5 TCPW 94 12.8 7.5 3.2 92.6 74.4 96.8 66.0 2.8 × 10 7 MWI 93 14.0 16.1 11.8 18.3 25.8 10.8 9.7 1.4 × 10 4 MWE 91 36.3 18.7 9.9 35.2 30.8 14.3 16.5

Result and Discussion  An Anti tibi biotic r otic res esis istance tance le leve vels of ls of En Ente terobact robacter eria iaceae ceae Percentage of antibiotic-resistant Number of total Number of Sampling Enterobacteriacea (%) heterotrophic bacteria Enterobacteriacea site (CFU/mL) isolates GTM LVX TMP AMP AMC CXM CTR 4.9 × 10 8 RCPW 79 13.9 10.1 16.5 57.0 39.2 67.1 57.0 2.7 × 10 5 TCPW 94 12.8 7.5 3.2 92.6 74.4 96.8 66.0 2.8 × 10 7 MWI 93 14.0 16.1 11.8 18.3 25.8 10.8 9.7 1.4 × 10 4 MWE 91 36.3 18.7 9.9 35.2 30.8 14.3 16.5 Removal efficiencies of total heterotrophic bacteria in both cephalosporin wastewater  treatment plant and municipal wastewater treatment plant were 3-log approximately

Result and Discussion  An Anti tibi biotic r otic res esis istance tance le leve vels of ls of En Ente terobact robacter eria iaceae ceae Percentage of antibiotic-resistant Number of total Number of Sampling Enterobacteriacea (%) heterotrophic bacteria Enterobacteriacea site (CFU/mL) isolates GTM LVX TMP AMP AMC CXM CTR 4.9 × 10 8 RCPW 79 13.9 10.1 16.5 57.0 39.2 67.1 57.0 2.7 × 10 5 TCPW 94 12.8 7.5 3.2 92.6 74.4 96.8 66.0 2.8 × 10 7 MWI 93 14.0 16.1 11.8 18.3 25.8 10.8 9.7 1.4 × 10 4 MWE 91 36.3 18.7 9.9 35.2 30.8 14.3 16.5 Removal efficiencies of total heterotrophic bacteria in both cephalosporin wastewater  treatment plant and municipal wastewater treatment plant were 3-log approximately Antibiotic resistant Enterobacteriaceae were popular in both cephalosporin producing  wastewater and municipal wastewater