5 12 Vol. 5 No. 12 2014 12 Journal of Food Safety and Quality Dec., 2014 张玫 #, 刘晓阳 #, 李开雄 *, 姬华 *, 卢士玲, 罗娟, 周红 (, 832000) 摘要 : 目的,, 方法 30, 8, 27% 8, 8 1 E27 1 E25 结果 8, 8 4 4 结论, E25, E27, E25 E27 关键词 : ; ; ; Study on viability of multiple drug-resistant and sensitive Escherichia coli in vitro gastrointestinal environment ZHANG Mei #, LIU Xiao-Yang #, LI Kai-Xiong *, JI Hua *, LU Shi-Ling, LUO Juan, ZHOU Hong (College of Food Science and Technology, Shihezi University, Shihezi 832000, China) ABSTRACT: Objective Escherichia coli is human and warm blooded animal intestinal important facultative anaerobic microflora group. It is indicator bacteria of water and food fecal pollution. The viability of multiple drug-resistant and drug sensitive Escherichia coli in vitro gastrointestinal environment was studied. The results provided a theoretical basis for control of harmful Escherichia coli. Methods The 30 retail food were 基金项目 : (31301469 31160329 31360392)(RCZX201225) (3152SPXY02033) (2013GA891006)(2013BA012) Fund: Supported by the National Natural Science Foundation of China (31301469, 31160329 and 31360392), the Scientific Research Foundation for High Level Talents of Shihezi University(RCZX201225), the Excellent Young Teachers Program of Shihezi University(3152SPXY02033), National Spark Plan Project (2013GA891006), and the Science and Technology Research Project of Xinjiang Production and Construction Corps(2013BA012) 通讯作者 :,,, E-mail: Lkx1956@126.com,,, E-mail: jihua229@126.com *Corresponding author: LI Kai-Xiong, Professor, College of Food Science and Technology, Shihezi University, BeiSi Road, Shihezi City, 832000, China. E-mail: Lkx1956@126.com JI Hua, Associate Professor, College of Food Science and Technology, Shihezi University, BeiSi Road, Shihezi City, 832000, China. E-mail: jihua229@126.com # # ZHANG Mei and LIU Xiao-Yang are co-first authors
12, : 4133 collected from supermarkets of Kuitun City, the 8 positive samples were identified. The contamination rate was 27%. Then the disk diffusion method was used to conduct the drug test for 8 E.coli strains isolated from 8 positive samples respectively. A multi-drug resistant E.coli E27 and a drug sensitive E.coli E25 were implemented in artificial gastric juice and the intestinal juice by tolerance test. Results The results show that the measured eight E.coli were sensitive to most drugs. The resistant bacteria were keeping resistance for ampicillin and tetracycline. There were four drug-resistant bacteria and four sensitive bacteria. Conclusion The tolerance was good for E.coli E25 in vitro gastrointestinal juice. And the tolerance was not high for E.coli E27 in vitro gastrointestinal juice. The decreasing trend of sensitive bacteria E25 was slower than multi-drug resistant bacteria E27. KEY WORDS: multiple drug-resistant; Escherichia coli; gastrointestinal environment; disc diffusion method 1 引言 (Escherichia coli, E.coli),,,, [1],,,,,,,, 2 材料与方法 2.1 材料与试剂 2.1.1 菌种 8,,,,,,, 30, 8 2.1.2 主要试剂 [ (10 μg/ ), (30 μg/ ), (30 μg/ ), (10 μg/ ), (10 μg/ ), (5 μg/ ), (5 μg/ ), (30 μg/ ), (30 μg/ ), (30 μg / ), (100 μg/ ), (SMZ/TMP: 23.75 μg/12.5 μg/ ), (15 μg/ ), (10 μg/ ), B(300 μg/ ), (10 μg/ ), (30 μg/ )]( ); LST EC EMB MH LB ( ) 2.1.3 仪器与设备 (Lab Tech, ); (SPX-250B-Z, ); (SW-CJ, ); (BCD-268E, ); (HPX-9272MBE, ) 2.2 实验方法 2.2.1 LST EC 培养基培养 25 g 225 ml 0.85%, 1 ml LST, 37 48 h, LST, 100 μl EC, 44 48 h [2] 2.2.2 EMB 培养基划线培养及生化鉴定 EC, EMB, 37 18~24 h, [3] LB, MR-VP 2.2.3 大肠杆菌的 PCR 检测 PCR [4] ATCC 25922 (1) : 25 μl: E colidna, 1 μl; UidA F, 1 μl; UidA R, 1 μl; 10 buffer, 2.5 μl;
4134 5 dntp, 2 μl; Taq, 0.5 μl;, 17 μl [5] (2) : (3) 1%,, Tris- (0.5 TBE) 1~2 mm DNA,,, 80 V [6] 2/3,,, DNA, UidA 2.2.4 多重耐药筛选实验 8, (Clinical and Laboratory Standards Institute, CLSI, NCCLS) [7], ATCC 25922 8 1.5 10 8 CFU/mL MH 20 ml,, 0.1 ml, 24 mm, 15 mm,,, 5 min, 36, 24 h :,,,, : a. (susceptible, S): b. (intermediate, I):, c. (resistant, R): 表 1 K-B 法药敏试验纸片解释结果 Table 1 Interpretation results of K-B disk method (mm) R I S 13 14~16 17 14 15~22 23 17 18~20 21 12 13~14 15 13 14~15 16 15 16~20 21 13 14~16 17 14 15~18 19 12 13~17 18 14 15~16 17 17 18~20 21 10 11~15 16 13 14~22 23 13 14~17 18 11 12~14 15 13 14~18 19 8 9~11 12
12, : 4135 1 17, 13, ; 14~16 ; 17, 2.2.5 体外胃肠道环境模拟试验, (1) [8], : : 0.85% NaCl, 121 20 min : 0.85 g NaCl 100 ml, HCl ph 2.5, 121 20 min,,, 1% : a b 2:1 a. : 1.1 g NaHCO 3, 0.85 g NaCl 100 ml, NaOH ph 6.8, 121 20 min, 1.5 g b. : 1.2 g 100 ml, 121 20 min, (2) E25 E27, (3 10 8 CFU/mL), (1 10 3 CFU/mL) 1 ml, 49 ml, 37, 30 min ( 0 h), 4 h 9, 10 [9] (3) 3 h, 1 ml 49 ml, 30 min ( 0 h), 4 h 9, 10 3 结果与分析 3.1 大肠杆菌分离鉴定结果 30, 8, : E13, E20, E21, E22, E23, E24, E25, E27 3.2 多重耐药大肠杆菌的筛选与结果 (K-B ),,, 2 [7] 2, 1, E13 E22 E25, 16 ; E20,, 14 ; E21, 15 ; E23,, 14 ; E24,, 15 ; E27,, 10 3 3, E20, E23 E24, E27,, E27 E21, E13, E22 E25, E25, E25 E25 E27 3.3 多重耐药与敏感型大肠杆菌在体外模拟胃肠道环境的耐受性研究 4 1 2,, E25 E27 2 h, E25 E27,, E27 E25 4 h, E25, E25 3.5 h, E27 3.5 h, E27
4136 5 表 2 17 种抗生素对所试菌株的抑菌直径 ( 单位 : mm) Table 2 The bacterial inhibition diameters of 17 antibiotics resisted strains (Unit: mm) E13 E20 E21 E22 E23 E24 E25 E27 17.33 7.00 16.20 18.67 22.50 18.17 21.50 9.00 31.33 30.50 26.10 30.83 34.50 31.00 32.33 36.00 27.33 27.00 22.47 27.17 30.50 24.50 29.17 29.50 23.33 25.83 19.63 21.67 25.50 21.33 25.83 23.33 30.67 28.67 32.17 29.33 35.00 28.00 34.33 38.00 34.67 38.00 37.73 29.67 30.00 23.67 35.00 38.50 31.33 33.67 30.93 25.00 28.67 20.00 35.17 31.50 24.67 28.00 23.83 27.67 8.00 8.00 24.33 7.00 26.33 19.83 26.60 26.50 25.50 26.17 27.33 10.83 20.67 22.17 20.07 20.33 22.25 20.33 24.17 22.17 27.33 28.17 23.63 24.83 26.50 25.67 29.67 20.17 30.00 31.00 26.97 32.83 28.33 28.50 33.00 7.00 20.00 18.00 22.67 15.00 14.67 16.00 22.83 18.17 22.67 9.00 24.83 21.67 29.00 20.00 30.33 14.50 20.00 20.67 22.67 16.17 16.17 15.50 17.67 16.00 30.67 30.33 29.40 30.33 16.50 26.00 31.17 24.67 17.67 14.17 14.63 15.00 16.83 14.17 16.50 14.67 : 7 mm 表 3 8 株所试菌株对 17 种抗生素的敏感性结果 Table 3 The 17 antibioticals susceptibility results of 8 strains E13 E20 E21 E22 E23 E24 E25 E27 S R I S S S S R S S S S R R S R S S S S S S S R S S S S S S S I S S S S S S S R I I I I I I I I S R S S S S S I S S S S S S S S S S S S I S S S
12, : 4137 Table 4 表 4 菌株在人工胃液和肠液中的存活数 (CFU/mL) The number of strains in artificial gastric juice and intestinal juice (CFU/mL) ph3.0 0 h 0.5 h 1 h 1.5 h 2 h 2.5 h 3 h 3.5 h 4 h E25 1530 1210 1025 895 770 705 650 600 580 E27 1170 805 470 325 250 215 170 150 140 ph8.0 0 h 0.5 h 1 h 1.5 h 2 h 2.5 h 3 h 3.5 h 4 h E25 1015 960 760 750 495 465 400 165 350 E27 165 85 35 15 10 10 10 0 0 Fig. 1 1 The inactivation curve of E25 and E27 strains in artificial gastric juice E27, E21, E13, E22 E25, E25 8 4 4 (3), E25 E27 2 h,, E27 E25 4 h, E25, E25 3.5 h, E27 3.5 h, E27 Fig. 2 2 The inactivation curve of E25 and E27 strains in artificial intestinal juice 4 结论与讨论 4.1 结论 (1) 30, 8, 27% (2) 8 E23 E24, E20, 4.2 讨论, [10],, [11], [12] Miszczycha [13] O157: H7 O26: H11 150 min, 0.2% 1.8%,,
4138 5 [14,15], 参考文献 [1],,,. [J]., 2014, 35(7): 364 367. Ji H, Zhang M, Lu SL, et al. Review on drug resistance and virulence characterization of food borne Escherichia coli [J]. Sci Technol Food Ind, 2014, 35(7): 364 367. [2]. [D]. :, 2013. Li H. Isolation and Identification of Enteropathogenic E.coli from animals in Panjin city of Liaoning province and its drug resistance[d]. Changchun: Jilin Agricultural University, 2013. [3]. [D]. :, 2008. Wu H. Study on separation, identification of E.coli and treatment for piglet diarrhea in Jining district[d]. Taian: Shandong Agricultural University, 2008. [4],,,. [J]., 2011, 11(1): 196 201. Zhi S, Xi ML, Liu GG, et al. Detection of Shaanxi part area of different foodborne Escherichia coli resistance [J]. J Chin Inst Food Sci Technol, 2011, 11(1): 196 201. [5]. [D]. :, 2010. Zhi S. Characterization of Escherichia coli from retail meats and ready to eat food in some districts of shanxi province [D]. Yangling: Northwest A F University, 2010. [6]. [J]., 2009, 30(6): 83 85. Huang YL. Research on the experiment technique of agarose gel electrophoresis [J]. J Zhanjiang Norm Coll, 2009, 30(6): 83 85. [7] Clinical and Laboratory Standards Institute (CLSI).Performance Standards for Antimicrobial Susceptibility Testing; seventeenth Informational Supplement. Wayne, PA: Clinical and Laboratory Standards Institute. [8]. [D]. :, 2012. Li SS. Antibiotic susceptibility of potentially probiotic Lactoballus strains[d]. Baoding: Agricultural University of Hebei Province, 2012. [9] GB/T 4789.2-2010 -[S]. GB/T 4789.2-2010 Microbiological examination of food hygiene-aerobic plate count [S]. [10] Deris JB, Kim M, Zhang ZG, et al. The innate growth bistability and fitness landscapes of antibiotic-resistant bacteria [J]. Science, 2013, 342: 1068 1077. [11],,,. [J]., 2011, 31(2): 24 28. Shao ML, Xu Y, Zhao YL, et al. growth characteristics and virulence analysis of amultidrug-resistant Listeria monocytogenes [J]. J Microbiol, 2011, 31(2): 24 28. [12]. [D]. :, 2005. Wang LP. Relationship between resistance and pathogenicity of streptococcus strains and the effect of the infection on the activity of liver drug-metabolizing enzymes [D]. Nanjing: Agricultural University of Nanjing, 2005. [13] Miszczycha SD, Thevenot J, Denis S, et al. Survival of Escherichia coli O26:H11 exceeds that of Escherichia coli O157:H7 as assessed by simulated human digestion of contaminated raw milk cheeses [J]. Inter J Food Microbio, 2014, 172: 40 48. [14] Mao Y, Doyle MP, Chen J. Role of colanic acid exopoly saccharide in the survival of enterohaemorrhagic Escherichia coli O157:H7 in simulated gastrointestinal fluids [J]. Orig Artic, 2006, 42: 642 627. [15] Alpert C, Scheel J, Engst W, et al. Adaptation of protein expression by Escherichia coli in the gastrointestinal tract of gnotobiotic mice [J]. Environ Microbiol, 2009, 11(4): 751 761. 作者简介 ( 责任编辑 : 杨翠娜 ) 张玫, 在读硕士研究生, 主要研究方向为食品微生物 E-mail: ameislife@yeah.net 刘晓阳, 本科生, 主要研究方向为食品微生物 E-mail: 268024449@qq.com 李开雄, 教授, 硕士生导师, 主要研究方向为畜产品质量与安全 E-mail: Lkx1956@126.com 姬华, 博士, 副教授, 主要研究方向为食品质量与安全 E-mail: jihua229@126.com