Detection of antibiotics resistance and distribution of resistance genes in Vibrio parahaemolyticus from cultured shrimp
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摘要: 文章采用琼脂稀释法检测从养殖患病对虾中分离的36株副溶血弧菌(Vibrio parahaemolyticus)对16种药物的耐药性,并用PCR法检测喹诺酮类耐药基因qnrA、qnrB、qnrS和qnrVC,酰胺醇类耐药基因cat、optrA、floR和cfr,四环霉素类耐药基因tetA、tetB和tetM,磺胺类耐药基因sul1、sul2和sul3,氨基糖苷类耐药基因strA、strB、aadA和aacA,利福霉素类耐药基因arr,β-内酰胺类耐药基因blaCARB和大环内酯类耐药基因erm的携带状况,分析其耐药表型和基因型之间的相关性。结果显示,36株副溶血弧菌对β-内酰胺类药物氨苄西林耐药率最高(88.9%),其次为磺胺类药物磺胺甲噁唑(66.7%),硫酸新霉素、庆大霉素、头孢曲松和美罗培南呈现100%敏感。多重耐药副溶血弧菌比例高达61.1% (22/36),其中1株对6类抗菌药耐药。喹诺酮类耐药基因qnrVC检出率最高达72.2% (26/36);其次为氨基糖苷类耐药基因srtB,检出率58.3% (21/36);大环内酯类、利福霉素类耐药基因均未检测到。耐药基因检出率与耐药表型没有表现出一一对应的关系,提示副溶血弧菌耐药的复杂性。Abstract: We used agar dilution method to detect the resistance of 36 strains of Vibrio parahaemolyticus to 16 drugs. And used PCR amplication and DNA sequencing to detect the antimicrobial resistance to quinolones (qnrA, qnrB, qnrS, qnrVC), phenicols (cat, optrA, floR, cfr), tetracyclines (tetA, tetB, tetM), sulfonamides (sul1, sul2, sul3), aminoglycosides (strA, strB, aadA, aacA), rifampicin (arr), β-lactams (carB) and macrolides (erm). The results indicate that the isolates exhibited high resistance to ampicillin (88.9%) and sulfamethoxazole (SMZ, 66.7%), all susceptible to neomycin sulfate, gentamicin, ceftriaxone and meropenem. In general, multi-drug resistance (MDR) was highly prevalent (61.1%), and one isolate was resistant to six antimicrobials. Furthermore, 72.2% and 58.3% of the isolates were primarily mediated by qnrVC and strB, respectively; and the macrolides and rifamycin resistant genes were not detected in all the isolates. Obvious mismatch was found between the antimicrobial resistance phenotypes and genotypes, revealing the complexity of resistance to V. parahaemolyticus.
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随着对环境、食品安全以及可持续发展的日益关注,绿色饲料添加剂的研究与应用成为研究的热点之一。用氨基酸和微量元素的螯合物替代无机盐具有促进鱼虾、畜禽生长、提高成活率和饲料转化率、降低生产成本的作用[1-5],在国外倍受青睐,近年来在国内也逐渐得到重视。铜是对虾血蓝蛋白及其他一些金属酶类的重要组成成分,是对虾维持正常的生理功能及生长发育必须的微量元素之一;但是作为一种重金属,当其含量过高时对对虾有毒害作用,并且其在虾体内的积累会影响产品品质,排入水体会造成环境污染[6-8]。因此,合理控制饲料中铜元素的含量对于对虾养殖和品质,以及环境保护具有重要的意义,而选用利用率高的铜原料是其中的解决方法之一。凡纳对虾目前已成为我国养殖的主要对虾品种,虽然有关于铜需求量的报道,但却未见有关螯合铜应用的报道。本文将以凡纳对虾为研究对象,通过对对虾生长、免疫因子及其在虾体内残留量的影响,比较硫酸铜和蛋氨酸铜的应用效果,探讨蛋氨酸铜在凡纳对虾养殖中应用的可行性。
1. 材料与方法
1.1 饲料制备
取粤海饲料有限公司的无铜商品饲料,在每公斤饲料中分别添加含铜元素为0,7.5,15,30,45 mg的硫酸铜和螯合蛋氨酸铜,制粒,晒干,冷藏备用。
1.2 试验动物
凡纳对虾(Litopenaeus vannamei)。
1.3 实验设计
实验在粤海饲料有限公司东山岛实验基地进行。把个体均匀的凡纳对虾养在不同的桶中,随机分成9组,记录初重(见表 1),每组3桶,桶的容积为300 L,每桶中养殖对虾30尾,其中一组为对照(空白)组。试验早期,依据对虾体重,按其体重的8%~10%投喂,每天早、中、晚各投喂1次,在试验过程中根据对虾的生长及采食状况,调整投饵量,以下一次投喂时无残饵为准,养殖周期为8周。在实验中期(第4周)称重,结束时称重,并采血、肌肉和肝胰脏,计算生长速度、成活率,测量肌肉和肝脏组织的铜含量以及血清中酚氧化酶和超氧化物歧化酶的活性。
表 1 蛋氨酸铜和硫酸对对虾生长速度的影响Table 1 Effect of Met-Cu and copper sulphate in the feed on shrimp growth rate铜剂型
dosage form of copper铜添加量/mg·kg-1
copper content in feed初始体重/ g
initial body weight中期体重/ g
medium-term body weight终期体重/ g
final body weight前期生长速度/g·d-1
prior period growth rate后期生长速度/g·d-1
later period growth rate平均生长速度/g·d-1
average growth rate成活率/%
survival rate对照
control0 16.97±1.00 126.89±4.42 171.55±7.62 0.0975±0.0012 0.0592±0.0038 0.0823±0.0013 95.6 蛋氨酸铜
Met-Cu7.5 16.94±0.28 132.75±7.54 180.43±7.16 0.1027±0.0055 0.0632± 0.0018 0.0871±0.0031 95.6 15 16.90±0.16 130.74±3.63 171.85±5.68 0.101± 0.0041 0.0538±0.0108 0.0816± 0.0025 97.8 30 16.99±0.56 131.03±6.15 176.52±15.32 0.1006±0.0054 0.0596±0.0123 0.0844±0.0067 96.7 45 17.19±0.40 134.08±4.90 184.19±13.70 0.1031± 0.0036 0.0660±0.0119 0.0889± 0.0056 95.6 硫酸铜
coppes sulphate7.5 16.67±0.40 129.73±8.33 182.21±15.85 0.0997±0.0070 0.0688±0.0090 0.0877±0.0078 96.7 15 16.72±0.07 129.76±3.61 168.47±1.90 0.1009±0.0049 0.0512±0.0048 0.0804±0.0012 96.7 30 16.48±0.27 133.18±0.67 188.47±1.70 0.1035*±0.0017 0.0725*±0.0026 0.0906*±0.0018 97.8 45 16.83±0.13 129.51±2.84 169.35±5.59 0.1005±0.0035 0.0527±0.0066 0.0808±0.0017 96.7 注:*表示此值显著高于其他各组(P<0.05)
Note: *denotes this value is higher than the others significantly(P<0.05)1.4 酚氧化酶(PO)和超氧化物歧化酶(SOD)
PO活力的测定参照Horowitz(1952)及Ashida(1971)的测定方法,以L-dopa为底物测定[9]。SOD活力的测定参照邓碧玉等改良的连苯三酚自氧化法[10]。
1.5 肌肉和肝胰脏铜含量的测定
参照国标GB17378.6-1998。
1.6 统计
利用SPSS统计软件中的ANOVA功能进行统计分析。
2. 结果
2.1 生长速度
如表 1所示,实验前期和后期,添加硫酸铜的各实验组对虾个体的平均生长速度分别在0.0997~0.1035 g · d-1和0.0512~0.0725 g · d-1之间,无论在实验前期还是后期,只有每kg饲料添加30 mg铜的实验组的生长速度显著高于其它各组,其它各组间差异不显著;而添加螯合铜的各实验组差异不显著,在实验前期和后期对虾个体的平均生长速度分别为0.1006~0.1031 g · d-1和0.0538~0.0660 g · d-1之间,同对照组间也无显著差异。
2.2 成活率
各实验组的成活率在95.6%~97.8%之间,各组差异不显著,见表 1。
2.3 肌肉和肝胰脏铜含量
如表 2,各实验组肌肉铜的含量为3.01~5.55 mg · kg-1,平均为(4.17±1.80)mg · kg-1,各组间差异不显著,但个体间差异非常大;对照组肝胰脏铜含量最低,为33.75 mg · kg-1,投喂硫酸铜添加量为45 mg · kg-1饲料的实验组最高,为210 mg · kg-1。肝胰脏铜含量大大高于肌肉。
表 2 铜在对虾组织中的分布Table 2 Distribution of copper in shrimp tissues铜剂型 dosage form of copper 铜添加量/mg·kg-1饵料
copper content in feed肝铜含量/mg·kg-1肝组织
copper content in liver肌肉铜含量/mg·kg-1肌肉
copper content in muscle对照 control 0 33.775 3.773±0.840 蛋氨酸铜
Met-Cu7.5 120.318 3.301±1.148 15 183.407 4.392±3.099 30 155.405 3.039±0.991 45 185.337 4.061±1.397 硫酸铜
copper sulphate7.5 51.013 4.376±1.281 15 158.494 3.013±1.601 30 156.358 4.655±2.022 45 210.358 5.552±3.020 2.4 PO和SOD活力
如图 1、图 2,除铜添加量为15 mg · kg-1饲料的实验组外,各实验组PO活力都高于对照组,当铜的添加量为30 mg · kg-1饲料时,PO活力最高,添加蛋氨酸铜和硫酸铜的实验组分别为12.8和9.8;血清SOD活力随着铜添加量的增加而升高,对照组最低为357,当添加量达到30 mg · kg-1饲料时,SOD活力最高,蛋氨酸铜组和硫酸铜组间无差异,都为507,而当铜添加量为45 mg · kg-1饲料时,反而降低,蛋氨酸铜组和硫酸铜组分别为443和486。
3. 讨论
微量元素是动物体必须的一大类营养元素,特别是作为某些酶或激素必须的组成成分,直接参与机体的几乎所有生理生化功能,对生命活动起着非常重要的作用,传统上使用的微量元素都为本元素的无机盐类,存在着适口性差、易吸潮、不易吸收、对维生素可能造成破坏的缺点,因此,欧美等发达国家从30年前就开始研究替代产品。研究表明,氨基酸复合矿物元素因其融多种氨基酸与微量元素于一体,具有氨基酸的鲜香味及诱食作用,并且氨基酸螯合盐有利于机体的吸收,能够提高生物体对微量元素的利用率[11],逐渐受到人们的重视。吕景才、赵元凤等利用蛋白下脚料如毛发、蹄脚、血粉等通过酸水解、碱中和、螯合等过程,制备了锌、锰、铜、钴等的氨基酸复合物,并以此作为微量元素添加到饲料中投喂鲤鱼和非鲫,同添加无机盐进行比较,他们发现添加复合微量元素的实验组生长速度显著高于添加无机盐的对照组,而饲料系数低于对照组,复合微量元素的添加量少于无机盐也能达到同样的效果[3-4]。阳会军等认为斑节对虾对蛋氨酸铜的吸收利用效率高于硫酸铜,并且能够促进斑节对虾的生长,减少铜的使用[2]。但就本实验结果来看,添加在饲料中喂养凡纳对虾时,蛋氨酸铜同硫酸铜相比没有优势,而当铜添加量为30 mg · kg-1饲料时,硫酸铜的促生长效果要好于蛋氨酸铜,这同Davis发现的凡纳对虾对饲料铜的最低需要量为32 mg · kg-1[12]接近。
酚氧化酶是甲壳动物的酚氧化酶原激活系统的产物,在识别异物、释放调理素促进血细胞的吞噬和包囊以及产生杀灭和排除异物的凝集素和溶菌酶等免疫功能方面发挥着重要的作用,同机体的免疫功能有直接的关系[13];而SOD具有消除自由基的功能,当SOD酶活力降低时,生物体内自由基积累过多,将导致代谢紊乱、正常生理功能失调,免疫及防御水平下降,容易引起疾病的发生[14]。维持适当水平的PO与SOD水平对于对虾对异物入侵迅速作出反应和维持虾体健康是非常重要的。因此,我们选这2个因子作为凡纳对虾的免疫指标。本研究发现,饲料铜的含量对对虾的血清SOD和PO活力都有一定影响,当每kg基础饲料中添加30 mg铜时最高,在此时,SOD的活力两者间没有差异,而添加蛋氨酸铜组的PO活力高于添加硫酸铜组;添加蛋氨酸铜与硫酸铜不影响两种酶活力的变化趋势。
总的说来,无论从生长速度、还是从免疫因子以及肌肉中铜的含量来说,在凡纳对虾饲料中添加蛋氨酸铜并不比添加硫酸铜具有优势,在添加浓度为30 mg · kg-1,添加硫酸铜的实验组生长速度反而显著高于添加蛋氨酸铜的实验组,与其他作者的结果不同,这可能与生物的种类有关,也可能与生物的生长环境有关,具体原因需要进行进一步的深入研究。
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表 1 抗菌药物稀释范围、质控菌株质控范围及药敏试验结果判定标准
Table 1 Dilution range of antibacterial drugs, quality control range of quality control strains and criteria for determination of drug susceptibility test results
抗菌药物Antimicrobial 稀释范围Dilution range/(μg·mL−1) MIC判定标准Judgment standard/(μg·mL−1) ATCC25922质控范围Quality control range/(μg·mL−1) S I R 环丙沙星 Ciprofloxacin 0.12~64 ≤1 2 ≥4 0.004~0.015 诺氟沙星 Norfloxacin 0.06~32 ≤4 8 ≥16 0.03~0.12 恩诺沙星 Enrofloxacin 0.06~32 ≤0.5 1~2 ≥4 0.03~0.12 氟苯尼考 Florfenicol 0.25~128 ≤2 4 ≥8 2~8 氯霉素 Chloramphenicol 0.25~128 ≤8 16 ≥32 2~8 土霉素 Oxytetracycline 0.12~64 ≤4 8 ≥16 0.5~4 多西环素 Doxycycline 0.12~64 ≤4 8 ≥16 0.5~2 磺胺甲噁唑 Sulfamethoxazole 5~2 560 ≤256 − ≥512 4~32 复方新诺明 Compound sulfamethoxazole 0.125/2.375~64/1 216 ≤2/38 − ≥4/76 ≤0.5/9.5 硫酸新霉素 Neomycin sulfate 0.06~32 ≤2 4 ≥8 0.5~4 庆大霉素 Gentamicin 0.06~32 ≤4 8 ≥16 0.25~1 红霉素 Ergomycin 0.5~512 ≤0.5 1~4 ≥8 2~8 头孢曲松 Ceftriaxone 0.12~64 ≤1 2 ≥4 0.03~0.12 氨苄西林 Ampicillin 0.25~128 ≤8 16 ≥32 2~8 美罗培南 Meropenem 0.125~64 ≤4 8 ≥16 0.008~0.06 利福平 Rifampin 1~512 ≤1 2 ≥4 4~16 注:−. 缺少相应的参考数据 Note: −. Lack of corresponding reference data 表 2 耐药基因引物序列
Table 2 Primers sequences of drug resistance genes
基因名称Gene name 引物序列 (5'–3')Primer sequence 退火温度Annealing temperature/℃ 产物长度Length of product/bp 参考文献Reference qnrVC136 TTCTCACATCAGGACTTGCGGAACAATGATTACCCCT 55 615 [23] qnrVC457 ATAAAACAGACCAGTTATATGTACTATTAAACVCTAATTGCTCTA 55 630 [23] qnrA ATTTCTCACGCCAGGATTTGGATCGGCAAAGGTTAGGTCA 55 516 [24] qnrB GATCGTGAAACGCAGAAAGGACGATGCCTGGTAGTTGTCC 50 469 [25] qnrS ACGACATTCGTCAACTGCAATAAATTGGCACCCTGTAGGC 52 417 [26] tetA TTTCGGGTTCGGGATGGTCAGGCAGAGCAAGTAGAGG 55 696 [23] tetM GTGGACAAAGGTACAACGAGCGGTAAAGTTCGTCACACAC 55 405 [23] tetB GTCGCGGCATCGGTCATTTTTTCGCCCCATTTAGTG 55 489 [23] Sul1 GTGACGGTGTTCGGCATTCTTCCGAGAAGGTGATTGCGCT 63 779 [23] Sul2 GCGCAGGCGCGTAAGCTGATCGAAGCGCAGCCGCAATTC 65 793 [27] Sul3 GCAACAGTTGGTGCTAAACGAGAAGCAGATGTGATTGATTTGGGAG 56 578 [23] strA TGGCAGGAGGAACAGGAGGAGGTCGATCAGACCCGTGC 54 546 [27] strB ATCGTCAAGGGATTGAAACCGGATCGTAGAACATATTGGC 52 509 [23] aadA GGAGAATGGCAGCGCAATGTTACTGCGCTGTACCAAT 55 269 [23] aacA CTTGGRTGACCTCGGGATCCGATGCTCTATGAGTGGCTAA 54 344 [23] erm CCCGAAAAATACGCAAAATTTCATCCCTGTTTACCCATTTATAAACG 55 589 [23] cat ACAACAGCAACGGTACTAGCCAACTTTCACCGATGCCAC 52 550 [28] optrA TTCTCACCCAGATATGCCCGGGATCCCGGCAAACT 60 1395 [29] floR CTGCTGATGGCTCCTTTCGCCGTGGCGTAACAGAT 57 650 [30] cfr GTGAAGCTCTAGCCAACCGTCGCAGCGTCAATATCAATCCC 55 746 [31] blaCARB GCTGAGAGCTCATGAAAAAGTTACGTAGGATCCTTAACTTTATTTGTAGTGC 55 852 [32] 表 3 36株副溶血弧菌耐药状况
Table 3 Antibiotics resistance status of 36 strains of V. parahaemolyticus
抗菌药物Antimicrobial 耐药菌株数Number of resistant strains 抗菌药物Antimicrobial 耐药菌株数Number of resistant strains 恩诺沙星 Enrofloxacin 2 (5.6%) 复方新诺明 Compound sulfamethoxazole 8 (22.2%) 诺氟沙星 Norfloxacin 6 (16.7%) 硫酸新霉素 Neomycin sulfate 0 (0) 环丙沙星 Ciprofloxacin 6 (16.7%) 庆大霉素 Gentamicin 0 (0) 氟苯尼考 Florfenicol 4 (11.1%) 红霉素 Erythromycin 23 (63.9%) 氯霉素 Chloramphenicol 2 (5.6%) 头孢曲松 Ceftriaxone 0 (0) 土霉素 Oxytetracycline 17 (47.2%) 氨苄西林 Ampicillin 32 (88.9%) 多西环素 Doxycycline 4 (11.1%) 美罗培南 Meropenem 0 (0) 磺胺甲噁唑 Sulfamethoxazole 24 (66.7%) 利福平 Rifampin 9 (25%) 表 4 副溶血弧菌耐药基因携带率
Table 4 Drug resistance gene carrier rate of V. parahaemolyticus
耐药基因Resistance gene 检出率Detection rate/% 耐药基因Resistance gene 检出率Detection rate/% 耐药基因Resistance gene 检出率Detection rate/% qnrVC 72.2 cfr 22.2 strA 44.4 qnrA 41.7 tetA 16.7 strB 58.3 qnrB 0 terB 47.2 aadA 33.3 qnrS 36.1 tetM 13.9 aacA 16.7 cat 0 sul1 0 erm 0 optrA 0 sul2 13.9 blaCARB 47.2 floR 11.1 sul3 0 arr 0 表 5 36株副溶血弧菌耐药表型与其携带基因型关系
Table 5 Relationship between resistant phenotype of 36 V. parahaemolyticus strains and their carrying genotypes
菌株Strain 耐药谱型 Resistance profile 携带耐药基因 Resistance gene 301 NOR-CIP-SXT-CFR-MER-SMZ-E-AMP blaCARB 302 NOR-CIP-SMZ-E-AMP qnrVC-blaCARB 331 NOR-CIP-SMZ-E-AMP blaCARB 333 SMZ-E-AMP blaCARB 335 SMZ-E-AMP qnrVC-tetM-blaCARB 337 OTC-SMZ-E-AMP qnrVC-blaCARB 338 OTC-SMZ-E-AMP qnrVC-blaCARB 340 OTC-DOX-SMZ-AMP-RA qnrVC-blaCARB 361 ENR-NOR-CIP-SMZ-AMP qnrVC-aadA-blaCARB 362 ENR-NOR-CIP-SMZ-E-AMP aadA-blaCARB 365 NOR-CIP-OTC-SMZ-E-AMP blaCARB 371 OTC-SMZ-SXT-E-AMP qnrVC-sul2-strA-strB-aadA-blaCARB 372 OTC-SMZ-SXT-E-AMP qnrVC-sul2-strA-strB-aadA-blaCARB 383 OTC-SMZ-SXT-E-AMP floR-sul2-strA-strB-blaCARB 384 OTC-SMZ-SXT-E-AMP sul2-strA-strB-blaCARB 388 FFC-OTC-DOX-SMZ-SXT-AMP-RA qnrVC-tetB-tetM-sul2-blaCARB 389 SMZ-AMP aadA-aacA-blaCARB 391 SMZ-E qnrS-strB 392 SMZ-E-AMP qnrVC-tetB-strB 393 OTC-SMZ-SXT-AMP qnrVC-tetB-tetM-strB 394 OTC qnrVC-cfr-tetB-strA-strB 395 AMP qnrVC-qnrA-qnrS-cfr-tetB-strB-aadA 396 OTC-E-AMP-RA tetB-strA-strB 397 AMP-RA qnrVC-qnrA-tetB-strA 398 E-AMP qnrVC-qnrA-qnrS-tetA-strA 399 E qnrVC-qnrA-qnrS-cfr-strB-aadA-aacA 400 E-AMP qnrA-qnrS-tetA-tetB-strA-strB-aadA-aacA 401 E-AMP qnrVC-qnrA-qnrS-cfr-tetA-tetB-strA-strB-aadA 402 AMP qnrVC-qnrA-qnrS-cfr-tetB-strA-strB-aadA-aacA 403 OTC-AMP qnrVCqnrA-qnrS-cfr-tetA-tetB-strA-strB-aadA-aacA 404 AMP-RA qnrVC-qnrA-qnrS-cfr-tetA-tetB-strA-strB-aadA 405 AMP qnrVC-qnrA-qnrS-cfr-tetA-tetB-strA-strB-aacA 406 OTC-SMZ-SXT-E-AMP-RA qnrVC-qnrA-floR-tetB-strA 407 FFC-C-OTC-DOX-SMZ-SXT-E-AMP-RA qnrVC-qnrA-qnrS-floR-tetB-tetM-strB 408 FFC-OTC-SMZ-RA qnrVC-qnrA-qnrS-tetB-strA-strB 409 FFC-C-OTC-DOX-SMZ-E-RA qnrVC-qnrA-qnrS-floR-tetB-tetM-strB 注:ENR. 恩诺沙星;NOR. 诺氟沙星;CIP. 环丙沙星;FFC. 氟苯尼考;C. 氯霉素;OTC. 土霉素;DOX. 多西环素;SMZ. 磺胺甲噁唑;SXT. 复方新诺明;E. 红霉素;AMP. 氨苄西林;RA. 利福平 Note: ENR. Enrofloxacin;NOR. Norfloxacin;CIP. Ciprofloxacin;FFC. Florfenicol;C. Chloramphenicol;OTC. Oxytetracycline;DOX. Doxycycline;SMZ. Sulfamethoxazole;SXT. Compound Sulfoxime;E. Erythromycin;AMP. Ampicillin;RA. Rifampicin -
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