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Acta Microbiologica Sinica 2017, 57(5): 681-689 DOI: 10.13343/j.cnki.wsxb.20160342 Research Article 研究报告 污水厂产超广谱 β 内酰胺酶大肠杆菌通过接合水平传递耐药性 李晴 1, 张红娜 1, 刘玉庆 2, 翟静 3*, 常维山 1 271000 2 250000 3 271000 1* 摘要 : β- 50 ESBLs 15 ESBLs 7 PCR 80 50 ESBLs 35 70% PCR bla TEM bla CTX-M bla SHV 1 关键词 : β- (Escherichia coli) 20 E. coli β- β (extended-spectrum β-lactamases ESBLs) β β 1 2 3 ESBLs 基金项目 :(SDA/T-11-09) * 通信作者 Fax +86-538-8248213 E-mail wschang@sdau.edu.cn Fax +86-538-6222053 E-mail jzhai@tsmc.edu.cn 收稿日期 :2016-08-31 修回日期 :2016-12-01 网络出版日期 :2016-12-21

682 Qing Li et al. Acta Microbiologica Sinica, 2017, 57(5) [1 2] R [3] ESBL [4] 1 材料和方法 1.1 25 30 25 1 0.5 m 20 ml 20 ml 4 C 24 h 1.2 0.45 μm 5 ml MEE (MEE broth) 37 C 12 h 37 C 12 h 5 ml LB 8 12 h 7 3 20 C 16S rrna PCR 1.3 ESBLs [5] MH (CTX) / (CTX/Clav) (CAZ) / (CAZ/Clav) 2 37 C 6 h 2 5 mm ESBLs 1.4 50 ESBLs J53 (filter mating method) [6] 5 μl 5 ml (64 μg/ml J53 ) (100 μg/ml ) LB 37 C 4 5 h OD 600 0.6 0.5 ml 4 ml LB Ф0.45 μm LB 37 C 4 5 h 1 ml LB 50 μl (64 μg/ml) (100 μg/ml) LB 37 C 12 16 h 3 [7] [8] 1 actamicro@im.ac.cn

, 2017, 57(5) 683 表 1. 接合子筛选判定标准 Table 1. The screening criteria of transconjugants Strains Sodium azide Cefotaxime Sodium azide and cefotaxime Donor strain S R Recipient strain R S Transconjugant + R: resistant; S: susceptible; : no bacteria growing; +: colony growth. 1.5 (64 μg/ml) (100 μg/ml) LB ERIC-PCR ERIC-PCR [9] ERIC-F 5 -ATGTAAGCTCCT GGGGATTCAC-3 ERIC-R 5 -AAGTAAGTGAC TGGGGTGAGCG-3 ERIC-PCR J53 LB ERIC-PCR J53 1.6 (CLSI) CLSI(M100-S22 2012) (WHO) Kirby-Bauer (FFC) (SXT) (AMP) (AZT) (KAN) (KF) (FEP) (NOR) (STR) (CIP) (IPM) (C) (E) (CN) (TE) 15 ( ) ATCC 25922 CL-SI2012 GenBank [10 12] 7 ( ) PCR 2 DNA PCR ( ) NCBI BLAST GenBank 2 结果和分析 2.1 16S rrna PCR BLAST 3 表 2. PCR 引物序列 Table 2. Squences of primers used for PCR Genes Primer sequences (5 3 ) Product length/bp bla SHV bla TEM bla CTX-M qnra qnrb qnrs OXA 表 3. F:GGGTTATTCTTATTTGTCGCT R:GGGTTAGCGTTGCCAGTG F:GAGACAATAACCCTGGTAA ATG R:AATGATTAATCAGTGAGGC F:AAGAAAAGTGAAAGCGAA R:GTGAAGTAAGTGACCAGAA TC F:TCAGCAAGAGGATTTCTCA R:GGCAGCACTATTACTCCCA F:ATGACGCCATTACTGTATAA R:GATCGCAATGTGTGAAGTTT F:ACCTTCACCGCTTGCACATT R:CCAGTGCTTCGAGAATCAGT F:CTGTTGTTTGGGTTTCGCAAG R:CTTGGCTTTTATGCTTGATC 大肠杆菌分离率 Table 3. Isolation rate of Escherichia coli 913 886 548 627 562 576 591 Sampling position Samples Isolate strain Separation rate/% Water inlet 25 25 100 Aeration tank 30 30 100 Outlet 25 15 60

684 Qing Li et al. Acta Microbiologica Sinica, 2017, 57(5) 2.2 ESBLs CLSI2009 ESBLs 80 50 ESBLs 62.5% 4 2.3 ERIC-PCR 50 ESBLs 35 70% ERIC-PCR 1 2.4 35 15 5 2 表 4. 产 ESBLs 大肠杆菌分离率 Table 4. Isolation rate of ESBLs producing Escherichia coli Sampling position Samples ESBLs producing strains Water inlet 25 22 88.0 Aeration tank 30 20 66.7 Outlet 25 8 32.0 Separation rate/% 5.7% 94.3% 48.6% 1 1 J53 (J53 15 ) 2.5 PCR 6 7 bla TEM bla CTX-M bla SHV 1 OXA qnra 23 qnrs 2 qnrb BLAST bla TEM-1 bla SHV-11 bla CTX-M-15 3 讨论 图 1. ERIC-PCR 电泳结果 Figure 1. The results of ERIC-PCR. M: DL2000 plus DNA marker; lane 1: negative control; lane 2: J53; lane 3 8: product of gene fragment. [13] β- β- ESBLs [14] ESBLs β- actamicro@im.ac.cn

, 2017, 57(5) 685 表 5. 35 株供体菌与其接合子耐药表型 Table 5. Drug resistance phenotypes of donor strains and transconjugants No. Transconjugant Donor strain A1 AMP-KF-STR AMP-KF-STR-C-FFC-CN-TE-KAN A2 AMP-AZT-KF-FEP-STR-E-TE SXT-AMP-AZT-KF-FEP-STR-C-AZT-CIP-CN-TE-NOR A4 SXT-AMP-KF-FEP-STR-C-TE AMP-KF-STR-C-CN-TE-KAN-FFC-IPM A8 SXT-AMP-AZT-KF-STR AMP-AZT-KF-NOR-FEP-E-CIP-IPM A10 SXT-AMP-AZT-KAN-KF-STR-CIP-C SXT-AMP-AZT-KAN-KF-CN-TE-FEP-E-IPM A11 SXT-AMP-AZT-KAN-KF-STR-E SXT-AMP-AZT-KF-E-TE A12 SXT-AMP-AZT-KF-STR-C-TE AMP-AZT-KF-TE-CN-NOR A13 SXT-AMP-AZT-KF-STR SXT-AMP-AZT-KF-CN-TE-NOR-KAN-C-FFC A14 SXT-AMP-AZT-KF-STR SXT-AMP-AZT-KF-STR-CN-TE-KAN-C-E-CIP-FFC A15 AMP-AZT-KF-STR SXT-AMP-AZT-KAN-KF-STR-CN-TE-NOR-C-E-CIP-FFC A17 SXT-AMP-AZT-KF-STR SXT-AMP-AZT-KF-CN-TE-FEP-C-AZT-E-IPM A20 SXT-AMP-AZT-KAN-KF-STR SXT-AMP-AZT-KF-STR-TE-FEP A21 AMP-AZT-KF-STR-E SXT-AMP-AZT-KF-STR-E-TE B1 SXT-AMP-AZT-KF-STR SXT-AMP-AZT-KF-CN-TE-IPM B3 AMP-AZT-KAN-KF-STR SXT-AMP-AZT-KAN-KF-STR-CN-TE B4 AMP-AZT-KF-FEP-STR AMP-AZT-KF-STR-NOR-IPM-KAN B5 AMP-AZT-KF-STR SXT-AMP-AZT-KF-STR-CN-NOR-CIP-IPM B6 SXT-AMP-AZT-KF-STR SXT-AMP-AZT-KF-STR-CN-FEP B7 AMP-AZT--KF-STR SXT-AMP-AZT-KF-STR-TE B8 AMP-AZT-KF-STR SXT-AMP-AZT-KF-STR-CN-TE-NOR-C-CIP-FFC B9 AMP-KF-FEP--STR SXT-AMP-AZT-KF-FEP-TE B10 AMP-AZT-KF-STR SXT-AMP-AZT-KF-CN-TE B11 AMP-AZT-KF-FEP--STR SXT-AMP-AZT-KF-FEP-STR-TE-E B12 AMP-AZT--KF-FEP-STR SXT-AMP-AZT-KF-FEP-STR-IPM-TE B14 AMP-KF-FEP-STR AMP-KF-STR-AZT-IPM B15 AMP-AZT-KF-STR SXT-AMP-AZT-KF-FEP-E-IPM B17 AMP-AZT-KF-STR SXT-AMP-AZT-KF-STR-TE-E-IPM B18 AMP-AZT-KF-FEP-STR SXT-AMP-AZT-KF-FEP-STR-CN-TE-E-IPM C1 AMP--KF-STR SXT-AMP-KF-AZT-STR-CN-TE-NOR-KAN-C-AZT-CIP-FFC-IPM C3 SXT-AMP-AZT-KF-FEP-STR SXT-AMP-AZT-KF-STR-TE-IPM C4 SXT-AMP-AZT-KF-FEP SXT-AMP-AZT-KF-FEP-IPM-TE C5 AMP-AZT-KF SXT-AMP-AZT-KF-CIP-IPM-TE C6 AMP-AZT-KF SXT-AMP-AZT-KF-E-IPM-TE-STR C7 AMP-AZT-KF SXT-AMP-AZT-KF-FEP-TE-IPM C8 AMP-AZT-KF SXT-AMP-AZT-KF-TE-STR-IPM ESBLs bla CTX-M bla SHV bla TEM OXA 图 2. 35 株供体菌与其接合子对 15 种抗菌药物多重耐药情况 Figure 2. Multiple drug resistance of 35 strains of donors and transconjugants to 15 kinds of antimicrobial agents. ESBLs [15] ESBLs [16] ESBLs

686 Qing Li et al. Acta Microbiologica Sinica, 2017, 57(5) 表 6. 35 株供体菌与其接合子耐药基因型 Table 6. The resistant genotype of 35 strains of donors and transconjugants Number Transconjugant Donor strain A1 bla TEM-135 --bla SHV-11 --bla CTX-M-15 bla TEM-135 --bla SHV-11 --bla CTX-M-15 --qnrs A2 bla TEM-1 --bla CTX-M-15 bla TEM-1 --bla SHV-11 --bla CTX-M-15 --qnrs A4 bla TEM-135 --bla SHV-11 --bla CTX-M-15 bla TEM-135 --bla SHV-11 --bla CTX-M-15 --qnrs A8 bla TEM-116 --bla SHV-11 --bla CTX-M-15 bla TEM-116 --bla SHV-11 --bla CTX-M-15 A10 bla TEM-1 --bla SHV-11 --bla CTX-M-15 bla TEM-1 --bla SHV-11 --bla CTX-M-15 A11 bla TEM-1 --bla SHV-11 --bla CTX-M-15 bla TEM-1 --bla SHV-11 --bla CTX-M-15 --qnrs A12 bla TEM-1 --bla SHV-11 --bla CTX-M-15 bla TEM-1 --bla SHV-11 --bla CTX-M-15 --qnrs A13 bla TEM-1 --bla SHV-11 --bla CTX-M-15 bla TEM-1 --bla SHV-11 --bla CTX-M-15 --qnrs A14 bla TEM-1 --bla SHV-40 --bla CTX-M-15 bla TEM-1 --bla SHV-40 --bla CTX-M-15 --qnrs A15 bla TEM-181 --bla SHV-11 --bla CTX-M-15 bla TEM-181 --bla SHV-11 --bla CTX-M-15 A17 bla TEM-181 --bla SHV-11 --bla CTX-M-55 bla TEM-181 --bla SHV-11 --bla CTX-M-55 A20 bla TEM-1 --bla SHV-11 --bla CTX-M-15 bla TEM-1 --bla SHV-11 --bla CTX-M-15 --qnrs A21 bla TEM-1 --bla SHV-11 --bla CTX-M-15 bla TEM-1 --bla SHV-11 --bla CTX-M-15 --qnrs B1 bla TEM-1 --bla SHV-11 --bla CTX-M-15 bla TEM-1 --bla SHV-11 --bla CTX-M-15 --qnrs B3 bla TEM-1 --bla SHV-56 --bla CTX-M-15 bla TEM-1 --bla SHV-6 --bla CTX-M-15 --qnrs B4 bla TEM-1 --bla SHV-11 --bla CTX-M-15 bla TEM-1 --bla SHV-11 --bla CTX-M-15 --qnrb B5 bla TEM-1 --bla SHV-11 --bla CTX-M-15 bla TEM-1 --bla SHV-11 --bla CTX-M-15 B6 bla TEM-181 --bla SHV-40 --bla CTX-M-55 bla TEM-181 --bla SHV-40 --bla CTX-M-55 --qnrs B7 bla TEM-1 --bla SHV-11 --bla CTX-M-15 bla TEM-1 --bla SHV-11 --bla CTX-M-15 --qnrs B8 bla TEM-1 --bla SHV-11 --bla CTX-M-15 bla TEM-1 --bla SHV-11 --bla CTX-M-15 --qnrs B9 bla TEM-1 --bla SHV-11 --bla CTX-M-15 bla TEM-1 --bla SHV-11 --bla CTX-M-15 --qnrs B10 bla TEM-1 --bla SHV-11 --bla CTX-M-15 bla TEM-1 --bla SHV-11 --bla CTX-M-15 --qnrs B11 bla TEM-1 --bla SHV-56 --bla CTX-M-15 bla TEM-1 --bla SHV-56 --bla CTX-M-15 --qnrs B12 bla TEM-1 --bla SHV-11 --bla CTX-M-15 bla TEM-1 --bla SHV-11 --bla CTX-M-15 --qnrs B14 bla TEM-1 --bla SHV-11 --bla CTX-M-15 bla TEM-1 --bla SHV-11 --bla CTX-M-15 B15 bla TEM-1 --bla SHV-11 --bla CTX-M-55 bla TEM-1 --bla SHV-11 --bla CTX-M-55 B17 bla TEM-1 --bla SHV-11 --bla CTX-M-15 bla TEM-1 --bla SHV-11 --bla CTX-M-15 --qnrs B18 bla TEM-1 --bla SHV-11 --bla CTX-M-15 bla TEM-1 --bla SHV-11 --bla CTX-M-15 --qnrs C1 bla TEM-1 --bla SHV-11 --bla CTX-M-55 bla TEM-1 --bla SHV-11 --bla CTX-M-55 C3 bla TEM-116 --bla SHV-11 --bla CTX-M-15 bla TEM-116 --bla SHV-11 --bla CTX-M-15 --qnrs C4 bla TEM-1 --bla SHV-79 --bla CTX-M-15 bla TEM-1 --bla SHV-79 --bla CTX-M-15 --qnrs C5 bla TEM-1 --bla SHV-11 --bla CTX-M-55 bla TEM-1 --bla SHV-11 --bla CTX-M-55 --qnrb C6 bla TEM-1 --bla SHV-11 --bla CTX-M-15 bla TEM-1 --bla SHV-11 --bla CTX-M-15 C7 bla TEM-1 --bla SHV-11 --bla CTX-M-15 bla TEM-1 --bla SHV-11 --bla CTX-M-15 --qnrs C8 bla TEM-1 --bla SHV-11 --bla CTX-M-15 bla TEM-1 --bla SHV-11 --bla CTX-M-15 actamicro@im.ac.cn

, 2017, 57(5) 687 表 7. 35 株供体菌与其接合子各耐药基因检出率 Table 7. The detection rate of resistance genes of 35 strains of donors and transconjugants Gene type Donor strain Transconjugant bla TEM 35 35 bla SHV 35 34 bla CTX-M 35 35 OXA 0 0 qnra 0 0 qnrb 2 0 qnrs 23 0 ESBLs 80 50 ESBLs ESBLs ESBLs 32% 88% ESBLs ESBLs [17] R J53 J53 50 ESBLs ERIC-PCR 35 70% 35 [18] 94.3% [19] 5.7% 48.6% 1 1 aada1 aada2 [20] ESBLs 100% ESBLs bla CTX-M bla SHV bla TEM OXA 5 50 ESBLs J53 35 ESBLs bla CTX-M bla SHV bla TEM 3 ESBLs OXA

688 Qing Li et al. Acta Microbiologica Sinica, 2017, 57(5) bla TEM bla CTX-M bla SHV 1 β- 3 bla CTX-M bla SHV bla TEM 3 bla CTX-M bla SHV bla TEM [1] Diwan V, Chandran SP, Tamhankar AJ, Stålsby Lundborg C, Macaden R. Identification of extended-spectrum β-lactamase and quinolone resistance genes in Escherichia coli isolated from hospital wastewater from central India. Journal of Antimicrobial Chemotherapy, 2012, 67(4): 857 859. [2] Jouini A, Vinue L, Slama KB, Sáenz Y, Klibi N, Hammami S, Boudabous A, Torres C. Characterization of CTX-M and SHV extended-spectrum β-lactamases and associated resistance genes in Escherichia coli strains of food samples in Tunisia. Journal of Antimicrobial Chemotherapy, 2007, 60(5): 1137 1141. [3] Mokracka J, Koczura R, Kaznowski A. Multiresistant enterobacteriaceae with class 1 and class 2 integrons in a municipal wastewater treatment plant. Water Research, 2012, 46(10): 3353 3363. [4] Wang HD, Xu GF, Jiao WW, Zhang XY. Investigation of antimicrobial agents resistance transferred horizontally in fluoroquinolone resistant Escherichia coli from swine. Acta Veterinaria et Zootechnica Sinica, 2016, 47(4): 805 811. (in Chinese),,,.., 2016, 47(4): 805 811. [5] Chen MJ. Bacterial resistance and detection method of beta-amide. Chinese Journal of Laboratory Medicine, 2001, 24(7): 197 200. (in Chinese). β-., 2001, 24(7): 197 200. [6] Clewell DB, An FY, White BA, Gawron-Burke C. Sex pheromones and plasmid transfer in Streptococcus faecalis: a pheromone, cam373, which is also excreted by Staphylococcus aureus//helinski D, Cohen SN, Clewell DB, Jackson DA, Hollaender A. Plasmids in Bacteria. New York: Plenum Press, 1985: 489 503. [7] An W, Zhang XY, Xu GF, Zhang L, Zhang YH, Li CY, Wu JD, Long ZC. Study of antibiotic resistance transferred horizontally in multidrug-resistant Escherichia coli from swine. Chinese Journal of Veterinary Medicine, 2014, 50(5): 76 78, 81. (in Chinese),,,,,,,.., 2014, 50(5): 76 78, 81. [8].., 2006. [9] Zhang H, Yang ZQ, Zhao J, Wei RC, Wang R. Generating of ERIC-PCR molecular typing method and its application in Escherichia coli. Jiangsu Journal of Agricultural Science, 2010, 26(5): 1098 1103. (in Chinese),,,,. ERIC-PCR., 2010, 26(5): 1098 1103. [10] Song XH, Yin YF, Huang YF, Liu XK. Detection of TEM, SHV and CTX-M genotype of ESBLs-producing gram-negative bacteria. Sichuan Journal of Physiological Sciences, 2011, 33(4): 154 156. (in Chinese),,,. ESBLs TEM SHV CTX-M., 2011, 33(4): 154 156. [11] Hu HY, Wang DG, Wei ZY, Zheng YW, Chen LM, Fang XF, Qi YX. Detection of multi-drug resistant Escherichia coli OXA gene and cluster analysis. Journal of Medical Research, 2012, 41(9): 129 133. (in Chinese),,,,,,. OXA., 2012, 41(9): 129 133. [12] Jiang Y, Zhou ZH, Qian Y, Wei Z, Yu Y, Hu S, Li L. Plasmid-mediated quinolone resistance determinants qnr and aac(6 )-Ib-cr in extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae in China. Journal of Antimicrobial Chemotherapy, 2008, 61(5): 1003 1006. [13] Lewis II JS, Herrera M, Wickes B, Patterson JE, Jorgensen JH. First report of the emergence of CTX-M-type extended-spectrum β-lactamases (ESBLs) as the pre-dominant ESBL isolated in a U.S. health care system. Antimicrobial Agents and Chemotherapy, 2007, 51(11): 4015 4021. [14] Cantón R, Novais A, Valverde A, Machado E, Peixe L, Baquero F, Coque TM. Prevalence and spread of extended-spectrum β-lactamase-producing Enterobacteriaceae in Europe. Clinical Microbiology and Infection, 2008, 14(S1): 144 153. [15] Koczura R, Mokracka J, Jabłońska L, Gozdecka E, Kubek M, Kaznowski A. Antimicrobial resistance of integron-harboring Escherichia coli isolates from clinical samples, wastewater treatment plant and river water. Science of the Total Environment, 2012, 414: 680 685. [16] Alexy R, Sommer A, Lange FT, Kümmerer K. Local use of antibiotics and their input and fate in a small sewage treatment plant-significance of balancing and analysis on a local scale vs. nationwide scale. Acta Hydrochimica et Hydrobiologica, 2006, 34(6): 587 592. actamicro@im.ac.cn

, 2017, 57(5) 689 [17] Diallo AA, Brugère H, Kérourédan M, Dupouy V, Toutain PL, Bousquet-Mélou A, Oswald E, Bibbal D. Persistence and prevalence of pathogenic and extended-spectrum beta-lactamase-producing Escherichia coli in municipal wastewater treatment plant receiving slaughterhouse wastewater. Water Research, 2013, 47(13): 4719 4729. [18] Cavaco LM, Hansen DS, Friis-Møller A, Aarestrup FM, Hasman H, Frimodt-Møller N. First detection of plasmid-mediated quinolone resistance (qnra and qnrs) in Escherichia coli strains isolated from humans in Scandinavia. Journal of Antimicrobial Chemotherapy, 2007, 59(4): 804 805. [19] Zhang H, Liu HC, He X, Chen LJ, Zhang TG. Gene location of integrons in Escherichia coli strain isolates from healthy human. Chinese Journal of Health Laboratory Technology, 2008, 18(6): 1004 1006. (in Chinese),,,,. I IntI1., 2008, 18(6): 1004 1006. [20] Zhao HX, Shen JZ, An XP, Fan HL, Cao JS, Li PF. Characterization of integrons in multiple antimicrobial resistant Escherichia coli isolates from bovine endometritis. Research in Veterinary Science, 2011, 91(3): 412 414. Transfer of antimicrobial resistant genes of ESBL-producing Escherichia coli recovered from a wastewater treatment plant Qing Li 1, Hongna Zhang 1, Yuqing Liu 2, Jing Zhai 3*, Weishan Chang 1* 1 College of Animal Sciences, Shandong Agricultural University, Tai an 271000, Shandong Province, China 2 Shandong Academy of Agricultural Sciences, Jinan 250000, Shandong Province, China 3 School of Basic Medical Sciences, Taishan Medical University, Tai an 271000, Shandong Province, China Abstract: [Objective] We explored the role of mobile plasmids in transferring antimicrobial resistant genes in extended-spectrum beta-lactamases (ESBLs)-producing Escherichia coli from a wastewater treatment plant. [Methods] Based on the conjugation experiments of ESBL-producing E. coli collected from wastewater, the disk diffusion assay and polymerase chain reaction (PCR) were used to determine the transfer of antimicrobial resistance. [Results] A total of 50 ESBL-producing E. coli were collected from 80 water samples (50/80, 62.5%), and successful conjugations were detected among 35 isolates (35/70, 70.0%). The results of PCR further showed that bla CTX-M and bla TEM genes were all capable of conjugation transfer, but the horizontal transfer of bla SHV gene was only detected in one isolate and resistance gene encoding fluoroquinolone was not capable of conjugation transfer in this study. [Conclusions] Plasmids carrying different antimicrobial resistant genes may exhibit different capacity of horizontal transfer. Importantly, the mobile plasmids played a very important role in the horizontal transfer of antimicrobial resistance genes of ESBL-producing E. coli. Keywords: extended-spectrum beta-lactamases, Escherichia coli, antimicrobial resistance genes, plasmid, conjugation, horizontal transfer ( 本文责编 : 张晓丽 ) Supported by the Shandong Province Agricultural Industry Technology Innovation System of Poultry Innovation Team (SDA/T-11-09) * Corresponding author. Weishan Chang, Fax: +86-538-8248213, E-mail: wschang@sdau.edu.cn; Jing Zhai, Fax: +86-538-6222053, E-mail: jzhai@tsmc.edu.cn Received: 31 August 2016; Revised: 1 December 2016; Published online: 21 December 2016