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1 4 5 Vol. 4 No Journal of Food Safety and Quality Oct., 213 谢 倩 1, 姚明 2, 刘媛 2, 刘宇贺 2, 李郁 2* (1., 2351; 2., 2336) 摘要 : 目的 方法 PCR, 1 ; K-B 结果 1%(1/1), 1%(1/1); 11 ; 14, 1%, 18.2%, 36.4%, 结论, 关键词 : ; ; ; ; Detection of Salmonella from the commercial egg shell and egg contents and analysis of its drug resistance in Hefei area XIE Qian 1, YAO Ming 2,LIU Yuan 2, LIU Yu-He 2, LI Yu 2* (1. Institute of Product Quality Supervision & Inspection of Anhui Province, Hefei 2351, China; 2. College of Animal Science and Technology, Anhui Agricultural University, Hefei 2336, China) ABSTRACT: Objective To understand the carrying rate of Salmonella and analyze of their serotype distribution and drug resistance from egg shell and egg content in commercial eggs. Methods A total of 1 eggs from market and supermarket in Hefei were detected by PCR technology and 14 antibiotics were tested by K-B method based on national standards. Results The detection rate of Salmonella from shell surface was 1% (1/1), and egg contents was 1% (1/1); the 11 isolated strains were identified as S. anatum, and they were all resistance to penicillin, 18.2% of them was resistance to streptomycin, 36.4% of them was medium resistance. Conclusion The carrying rate of Salmonella of egg shell surface was higher than that of egg content from commercial eggs, for antibiotic selection pressure could lead to the difference of the serotype and drug resistance of Salmonella isolates. KEY WORDS: egg; egg shell surface; egg contents; Salmonella; drug resistance 基金项目 : (29BADB9B1) (KJ28B56) Fund: Supported by the National science and technology support project (29BADB9B1), Colleges and universities in anhui province provincial natural science research project (KJ28B56) and Poultry industry system fund in anhui province * 通讯作者 :,,, [email protected] *Corresponding author: LI Yu, Professor, College of Animal Science and Technology, Anhui Agricultural University, No.13, Changjiang West Road, Hefei 2336, China [email protected]

2 引言,,,,,,,, [1] 4.2%,,, (PCR),, K-B β-, 2 材料与方法 2.1 材料 样品 ( A B C I, J), 1, 参考菌株 质控菌株 (ATCC25923) (ATCC25922) 主要试剂 (BPW) (TTB) (SC) (BS) (XLD) (TSI) ; 1 PCR buffer dntps(2.5 mol/l) Taq DNA (5 U/µL) 6 Loading Buffer DNA Marker- DL2 DNA Marker-DL3 DNA 2 Taq PCR MasterMix ( ) (59 ) 14 ( PG AMO GEN CIP TET SXT ENO FR STR DOX AMP FFC KAN CRO) MH MH 2.2 方法 沙门氏菌的分离培养 蛋壳表面沙门氏菌的分离培养, BPW 18 h, TTB SC, 18~24 h, BS XLD, 2 TSI, 37 18~24 h, PCR 蛋内容物沙门氏菌的分离培养,, 25 ml BPW, 沙门氏菌分离株的 PCR 鉴定 引物的选择 PCR [2] : P1: 5 -ACTGGCGTTATCCCTTTCTCTGGTG-3 P2: 5 -ATGTTGTCCTGCCCCTGGTAAGAGA 沙门氏菌总 DNA 提取 ( 热裂解法 ) 18 h, 2 μl 1.5 ml, 5 r/min 3 min,, 3 μl ddh 2 O TE (ph 8.), 1 min, 1 min, 12 r/min 3 min, DNA 沙门氏菌 PCR 扩增 25 μl, Buffer MIX(MgCl 2 contained) 3 μl, dntps(2.5 mmol/l) 2 μl, P1 P μl, Taq DNA (5 U/μL).25 μl, DNA 3. μl, ddh 2 O μl

3 第5期 谢 157 倩, 等: 合肥市售鸡蛋壳表面与内容物沙门氏菌污染的检测及其分离株耐药性分析 反应增条件为 94 5 min; 94 3 s, 6 3 s, s, 共 35 个循环; 72 1 min 沙门氏菌 PCR 产物分析 取 7 μl PCR 产物 经 1%的琼脂糖凝胶(含.5 mg/l 溴化乙锭)电泳, 在 凝胶成像仪中观察结果并拍照 预期扩增的沙门氏菌 特异性条带为 495 bp 沙门氏菌分离株的血清型鉴定 [3] Fig. 1 按照 GB/T 执行 沙门氏菌分离株的药敏试验 图 1 沙门氏菌的 PCR 扩增电泳图 Amplification electrophoresis figure of PCR for Salmonella isolates 注: 为样品中沙门氏菌阴性; 按照 WHO 推荐的 K-B 琼脂纸片扩散法进行, 根 为样品中沙门氏菌阳性; 11 为 DNA Marker DL2; 据美国临床试验室标准委员会(CLSI NCCLS)29 13 为沙门氏菌阳性对照 [4] 版执行标准判定结果 沙门氏菌分离株的血清型鉴定 结果 11 株沙门氏菌分离株均为鸭沙门氏菌, 见表 合肥市售鸡蛋蛋壳表面及其内容物沙门氏 菌的检出情况 沙门氏菌分离株对药物的感受性 3.3 在对 14 种抗生素的药敏试验中, 11 株鸭沙门氏 在被检的 1 份市售鸡蛋样品中, 共检出沙门氏 菌分离株对青霉素 1%耐受; 对链霉素的耐药率为 菌 11 株, 总检出率为 1%, 其中蛋壳表面检出沙门氏菌 18.2%, 中等耐药率 36.4%; 而对阿莫西林 庆大霉 1 株(编号为 H1 H2 H3 I1 I2 I3 I4 I5 I6 素 环丙沙星 四环素 复方新诺明 恩诺沙星 痢 I7), 检出率为 1, 蛋内容物检出沙门氏菌 1 株(编号 特灵 强力霉素 氨苄青霉素 卡那霉素 头孢曲松 为 J1), 检出率为 1% 见表 1, PCR 扩增电泳结果见图 1 钠 12 种抗生素均 1%敏感, 见表 3 Table 1 表 1 蛋壳表面与蛋内容物中沙门氏菌检出情况 The situation of Salmonella detection for egg shell and egg content 蛋壳表面检出沙门氏菌 采样地点 蛋内容物检出沙门氏菌 采样数(枚) 检出数量(株) 检出率(%) 检出数量(株) 检出率(%) A 1 B 1 C 1 D 1 E 1 F 1 G 1 H I J 合计 Table 2 表 2 11 株沙门氏菌分离株的血清型分布 The serotype distribution for 11 strain of Salmonella isolates 菌株编号 O 抗原 H1 H2 H3 I1 I2 I3 I4 I5 I6 I7 J1 3, 1 H 抗原 1相 2相 e, h 6 判定结果 鸭沙门氏菌

4 158 4 Table 3 表 3 11 株鸭沙门氏菌分离株对 14 种抗生素的感受性分布 The susceptibility distribution of 14 antibiotics for 11 strains of S. anatum isolates (%) (%) (%) (SXT) 1 ( GEN) 1 (AMP) 1 (ENO) 1 (FR) 1 (STR) 45.4(5/11) 36.4(4/11) 18.2(2/11) (DOX) 1 (AMO) 1 (CIP) 1 (P-G) 1 ( KAN) 1 (FFC) 1 ( TET) 1 (CRO) 1 4 讨论 4.1 合肥市售鸡蛋沙门氏菌的污染状况 1, 1%; 1%,, [5] 4,, 1.67%~8.3%, 1.67%~5.% [6], 2%, [7] 15%, 3.3%,.2%~.4%,, [8], 25~65, 3~5, 2~28,,,,, ( ),,,,, 25 3 [6], [9-11] 1%,,,,,

5 5, : 合肥市售鸡蛋沙门氏菌分离株的血清型 [12] : 7.5%(6/81), 3.75%(3/81),,, 合肥市售鸡蛋沙门氏菌分离株的耐药性, 11 1% 18.2%, 36.4%, [13] 89,, 43.8%,, 3.3% 22.5% 22.5% 2.2% 2.2% 19.1% 19.1%;, 1.1% 9.% 7.9% 7.9% 6.7% 6.7% 2.2%, 5%,,,,, 参考文献 [1],,,. [J]., 211, 7(23): Liu JJ, Zhang HH, Fang YH, et al. Investigation on the carrying rate of Salmonella from nonsymptomatic chickens in Hefei and analysis of their serotype and genotype [J]. Chin J Microecol, 211, 7(23): [2],,,. [J]., 22, 23(3): 4 7. Huang JL, Jiao XA, Wen QY, et al. Application of polymerase chain reaction to rapid detection of Salmonella [J]. J Yangzhou Univ, 22, 23(3): 4 7. [3] GB [S]. GB National food safety standard Food microbiological examination: Salmonella [S]. [4] Wayne PA. Performance standards for antimicrobial susceptibility testing [J]. Ninth informational supplement NCCLS document M1-S9. National Committee for Clinical Laboratory Standards, 28. [5],,. [J]., 28, 35(11): Liu JM, Sun BZ, Jia YM. The analysis of microbial change in Different brand sold fresh egg storage [J]. China Anim Husbandry Vet, 28, 35(11): [6],,,. [J]., 28, 3: Liu MY, Wang YX, Kong DJ, et al. The analysis of microbial pollution in egg shell and content [J]. Meat Res, 28, 3: [7],,,. [J]., 21, 26(2): Zhao L, Luo HX, Huang YF, et al. The sold eggs detection and analysis of three main pathogenic microorganism [J]. Mod Food Sci Technol, 21, 26(2): [8],,. [J]., 28, 29(6): Liu H, Wang FS, Chu J. Some properties egg white lysozyme and the factors affecting the enzyme activity of study [J]. Chin J Biochem Drugs, 28, 29(6): [9] Messens W, Grijspeerdt K, Herman L. Eggshell penetration by Salmonella: a review [J]. World Poult Sci J, 25, 61: [1],,,. [J]., 211, 4: Hu Y, Zhu CH, Shan YJ, et al. Enteritis of Salmonella contamination of eggs [J]. Poul China, 211, 4: [11],,,. [J]. ( ), 211, 11: Duan ZY, Qin YH, Liu YR, et al. Egg shell detect Salmonella contamination on the surface before and after cleaning [J]. Agr Prod Proces, 211, 11: [12],. [J]., 2, 12(6): Zhang HL, Wei XY. Salmonella serotype distribution in anhui province [J]. Shanghai J Prev Med, 2, 12 (6): [13],,,. [J]., 29, 8: Wang JH, He C, Zha ZL, et al. Poultry Salmonella serotype distribution and drug resistance analysis [J]. YangQin Poul Dis Prev Control, 29, 8: ( 责任编辑 : 赵静 )

6 151 4 作者简介 谢倩, 本科, 高级工程师, 主要研究方向为食品卫生检验 [email protected] 李郁, 教授, 硕士生导师, 主要研究方向为动物传染病与兽医公共卫生学 [email protected] 生物毒素研究 专题征稿 投稿方式 : [email protected] 食品安全质量检测学报 编辑部