Isolation, identification and biological characteristics of lactobacillus from grass carp
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摘要: 随着水产养殖病害问题日益严重，抗生素和药物使用对环境造成的危害越来越大，开展绿色生态防控技术研究是实现水产健康养殖的重要途径。该研究从健康草鱼 (Ctenopharyngodon idella) 肠道分离获得8株乳酸菌 (Lactobacillus)，经生化反应和16S rRNA鉴定，均为植物乳杆菌 (L. plantarum)。生物学特性评价结果表明植物乳杆菌分离株Y190430的发酵性能最好，且具有良好的抗生素敏感性；与植物乳杆菌标准株ATCC8014和商品乳酸菌相比较，分离株Y190430对酸、碱、盐、温度等环境胁迫具有更好的耐受性，同时具有更快的产酸速率，并可通过代谢产物抑制病原菌生长，体外抑菌试验表明分离株Y190430对常见水产病原菌的拮抗能力更强。Abstract: As the problem of aquaculture diseases is growing, the harm of using antibiotics and drugs is more and more serious so the research on ecological control technology is an important way to achieve healthy aquaculture. In this study, eight lactobacillus strains were isolated from the intestinal tract of healthy grass carps (Ctenopharyngodon idella). The identification result indicates that all the eight strains were Lactobacillus plantarum. The results of biological characteristics evaluation show that L. plantarum strain Y190430 had a high growth rate, and it was sensitive to common antibiotics. In addition, compared with the standard strain and commercial lactobacillus, L. plantarum Y190430 represented better tolerance to environmental stress such as acid, alkali, salt and temperature. Besides, it had a higher acid production rate and inhibited the growth of pathogenic bacterias better. The results provide references for the research on ecological control of aquatic diseases.
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Key words:
- Lactobacillus /
- Fermenting /
- Antibiotic sensitivity /
- Ecological prevention and control /
- Probiotics
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图 1 分离菌株的菌落形态特征 (a) 及革兰氏染色 (b)
Figure 1. Colony morphological characteristics (a) and gram staining (b) of isolated strains
图 2 分离菌株的16S rRNA序列的PCR扩增结果
Figure 2. PCR amplification results of 16S rRNA sequence of isolated strains
M. DL2 000 Marker; 1. C13; 2. C14; 3. C15; 4. C16; 5. C17; 6. C18; 7. C20; 8. C35.
图 3 基于分离菌株16S rRNA序列的系统发育树
Figure 3. Phylogenetic tree based on 16S rRNA sequences of isolated strains
图 5 分离菌株Y190430的环境耐受性分析结果
a. 温度耐受性；b. 酸碱耐受性；c. 渗透压耐受性。
Figure 5. Environmental tolerance analysis results of Y190430 strain
a. Temperature tolerance; b. Acid base tolerance; c. Osmotic pressure tolerance.
图 6 分离株Y190430的产酸性能
a. 与标准株相比较具有显著性差异；b. 与商品株相比较具有显著性差异；ab. 与标准株、商品株相比较均具有显著性差异。
Figure 6. Ability to produce acid of Y190430 strain
a. Compared with standard strain, there is a significant difference; b. Compared with commercial lactobacillus, there is a significant difference; ab. Compared with standard strain and commercial lactobacillus, there is a significant difference.
表 2 分离菌株的生理生化特征
Table 2. Physiological and biochemical characteristics of isolated strains
生化项目
Biochemical item分离菌株
Isolated strainC13 C14 C15 C16 C17 C18 C20 C35 苦杏仁苷 Amy gdalin + + + + + + + + 阿拉伯糖 Pectinose + + + + + + + + 纤维二糖 Cellobiose + + + + + + + + 七叶苷 Aesculin + + + + + + + + 果糖 Fructose + + + + + + + + 半乳糖 Galactose + + + + + + + + 葡萄糖 Glucose + + + + + + + + 葡萄糖酸盐 Gluconic acid − − − − − − − − 乳糖 Lactose + + + + + + + + 麦芽糖 Maltose + + + + + + + + 甘露醇 Mannitol + + + + + + + + 甘露糖 Mannose + + + + + + + + 松三糖 Melezitose + + + + + + + + 蜜二糖 Melibiose + + + + + + + + 棉籽糖 Raffinose − − − − − − − − 鼠李糖 Rhamnose − − − − − − − − 核糖 Ribose + + + + + + + + 水杨素 Salicin + + + + + + + + 山梨糖 Sorbose − − − − − − − − 蔗糖 Sucrose + + + + + + + + 蕈糖 Trehalose + + + + + + + + 木糖 Xylose − − − − − − − − 注：+. 阳性；−. 阴性。 Note: +. Positive; −. Negative. 
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表 3 分离菌株Y190430对酸碱的耐受性
Table 3. Acid base tolerance of Y190430 strain
$ \overline { \boldsymbol{X}}\pm { \bf {SD}}$ 培养pH
Incubation pH菌落总数 CFU/(logCFU·mL−1) 植物乳杆菌Y190430
L. plantarum Y190430植物乳杆菌ATCC8014
L. plantarum ATCC8014商品乳酸菌
Commercial lactobacillus2.0 2.50±0.08AB 1.10±0.03 1.70±0.05 3.0 3.50±0.05AB 2.90±0.02 3.20±0.05 4.0 7.30±0.04AB 5.90±0.04 7.00±0.07 5.0 8.30±0.02AB 8.10±0.07 8.10±0.07 5.6 8.90±0.24 8.80±0.02 8.80±0.07 6.0 8.80±0.04AB 8.50±0.04 8.60±0.07 7.0 8.50±0.02A 8.30±0.02 8.40±0.08 8.0 8.20±0.04B 8.10±0.07 8.00±0.07 9.0 8.00±0.04AB 7.50±0.02 7.70±0.02 注：A. 植物乳杆菌Y190430与植物乳杆菌ATCC8014相比较具有显著性差异；B. Y190340与商品株相比较具有显著性差异。 Note: A. Significant difference between L. plantarum Y190430 and L. plantarum ATCC8014; B. Significant difference between L. plantarum Y190430 and commercial lactobacillus.
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表 4 分离菌株Y190430对渗透压的耐受性
Table 4. Osmotic pressure tolerance of Y190430 strain
$ \overline { \boldsymbol{X}}\pm { \bf {SD}}$ 氯化钠
质量分数
NaCl mass fraction/%菌落总数 CFU/(log CFU·mL−1) 植物乳杆菌Y190430
L. plantarum Y190430植物乳杆菌ATCC8014
L. plantarum ATCC8014商品乳酸菌
Commercial lactobacillus0.9 8.90±0.08B 8.80±0.08 8.50±0.16 2.0 8.70±0.16B 8.50±0.16 7.90±0.16 4.0 7.10±0.16 6.90±0.08 6.90±0.16 6.0 6.50±0.08 6.70±0.16 6.40±0.16 8.0 5.10±0.24 5.00±0.16 4.70±0.20 注：B表示植物乳杆菌Y190430与商品株相比具有显著性差异 (P<0.05)。 Note: B represents significant difference between L. plantarum Y190430 and commercial lactobacillus (P<0.05).
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表 5 分离株Y190430的药敏测试结果
Table 5. The results of antibiotic sensitivity of Y190430 strain
抗生素
Antibiotics标准抑菌直径
Standard inhibition zone diameter/mm植物乳杆菌 Y190430
L. plantarum Y190430耐药 (R)
Resistance中等敏感 (I)
Intermediate敏感 (S)
Susceptible抑菌圈直径
Inhibition zone diameter/mm敏感性
Susceptibility青霉素 Penicillin ≤19 20~27 ≥28 25.85±0.11 I 氨苄西林 Ampicillin ≤28 — ≥29 34.03±0.09 S 多西环 Doxycycline ≤12 13~15 ≥16 21.26±0.07 S 四环素 Tetracycline ≤14 15~18 ≥19 18.37±0.11 I 氯霉素 Chloramphenicol ≤12 13~17 ≥18 27.62±0.10 S 氟苯尼考 Florfenicol ≤12 13~17 ≥18 30.95±0.08 S 红霉素 Erythromycin ≤13 14~22 ≥23 25.71±0.05 S 林可霉素 Lincomycin ≤14 15~20 ≥21 28.25±0.07 S
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表 1 分离菌株的菌落形态特征
Table 1. Colony morphological characteristics of isolated strains
编号
No.是否光滑
Smooth or
rough边缘形状
Edge
shape菌落颜色
Strain
colour透明度
Transparency菌落形状
Strain
shapeC13 是 整齐 白色 无 圆形 C14 是 整齐 白色 无 圆形 C15 是 整齐 白色 无 圆形 C16 是 整齐 白色 无 圆形 C17 是 整齐 白色 无 圆形 C18 是 整齐 白色 无 圆形 C20 是 整齐 白色 无 圆形 C35 是 整齐 白色 无 圆形
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表 6 分离菌株Y190340对水产养殖常见致病菌的抑制作用
Table 6. Inhibitory effect of Y190340 strain on common pathogenic bacteria in aquaculture
致病菌
Pathogen平均抑菌圈直径 Average inhibition zone diameter/mm 植物乳杆菌 Y190430
L. plantarum Y190430植物乳杆菌 ATCC8014
L. plantarum ATCC8014商品乳酸菌
Commercial lactobacillus舒伯特气单胞菌 A. schubertii 8.11±0.02 8.53±0.02 8.32±0.06 嗜水气单胞菌 A. hydrophila 8.89±0.07A 7.69±0.08 9.76±0.05 迟缓爱德华氏菌 E. tarda 9.03±007AB 8.51±0.07 7.96±0.04 铜绿假单胞菌 P. aeruginosa 12.77±0.05B 10.32±0.06 11.43±0.04 维氏气单胞菌 A. veronii 9.89±0.02 7.53±0.07 9.76±0.07 植物乳杆菌 ATCC8014 L. plantarum ATCC8014 — — 乳酸乳球菌 NZ9000 L. lactis NZ9000 — — — 注：—. 无抑菌作用；A. 与标准株相比较具有显著性差异；B. 与商品株相比较具有显著性差异；AB. 与标准株、商品株相比较均具有显著性差异。 Note: —. No bacteriostatic effect; A. Compared with the standard strain, there is a significant difference; B. Compared with commercial lactobacillus, there is a significant difference; AB. Compared with the standard strain and commercial lactobacillus, there is a significant difference.
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[1] WANG P, JI J, ZHANG Y. Aquaculture extension system in China: development, challenges, and prospects[J]. Aquacult Rep, 2020, 17: 1-9. [2] 农业农村部渔业渔政管理局. 2020中国渔业统计年鉴[M]. 北京: 中国农业出版社, 2020: 1, 24. [3] 朱文根, 李星浩, 饶刘瑜, 等. 感染草鱼呼肠孤病毒对肠道菌群多样性的影响[J]. 水生生物学报, 2019, 43(1): 109-116. doi: 10.7541/2019.014 [4] 祖国掌, 余为一, 李槿年, 等. 草鱼细菌性败血症的诊断及流行病学调查[J]. 淡水渔业, 2000, 30(5): 35-37. doi: 10.3969/j.issn.1000-6907.2000.05.017 [5] BRUDESETH B E, WIULSRØD R, FREDRIKSEN B N, et al. Status and future perspectives of vaccines for industrialised fin-fish farming[J]. Fish Shellfish Immunol, 2013, 35(6): 1759-1768. doi: 10.1016/j.fsi.2013.05.029 [6] WANG Q C, JI W, XU Z. Current use and development of fish vaccines in China[J]. Fish Shellfish Immunol, 2020, 96: 223-234. doi: 10.1016/j.fsi.2019.12.010 [7] 马丽, 吴金英, 高凇泽, 等. 海豚链球菌simA和pgmA真核表达质粒对尼罗罗非鱼免疫保护的研究[J]. 南方水产科学, 2020, 16(3): 38-46. doi: 10.12131/20190163 [8] 徐吟梅. 首个草鱼出血病活疫水产疫苗成功问世[J]. 现代渔业信息, 2011, 26(3): 73-74. [9] 巩华. 水产疫苗添新军——嗜水气单胞菌败血症灭活疫苗获生产批文[J]. 海洋与渔业, 2012(3): 49-49. doi: 10.3969/j.issn.1672-4046.2012.03.045 [10] 钱冬, 陈月英, 沈锦玉, 等. 引起鱼类暴发性流行病的嗜水气单胞菌的血清型, 毒力及溶血性[J]. 微生物学报, 1995, 35(6): 460-464. [11] 魏文娟, 赵姝, 王元, 等. 副溶血弧菌养殖对虾分离株耐药性及耐药基因分析[J]. 南方水产科学, 2020, 16(1): 9-16. doi: 10.12131/20190165 [12] 郭秀平, 刘毅辉, 潘厚军, 等. 我国水产药物残留与休药期研究现状概述[J]. 海洋与渔业, 2018(11): 76-78. [13] ZIELIŃSKA D, DŁUGOSZ E, ZAWISTOWSKA-DENIZIAK A. Functional properties of food origin lactobacillus in the gastrointestinal ecosystem: in vitro study[J]. Probiotics Antimicrob Proteins, 2019, 11(3): 820-829. doi: 10.1007/s12602-018-9458-z [14] HYRONIMUS B, LE MARREC C, SASSI A H, et al. Acid and bile tolerance of spore-forming lactic acid bacteria[J]. Int J Food Microbiol, 2000, 61(2/3): 193-197. [15] MIKULSKI D, JANKOWSKI J, MIKULSKA M, et al. Effects of dietary probiotic (Pediococcus acidilactici) supplementation on productive performance, egg quality, and body composition in laying hens fed diets varying in energy density[J]. Poult Sci, 2020, 99(4): 2275-2285. doi: 10.1016/j.psj.2019.11.046 [16] KEWCHAROEN W, SRISAPOOME P. Probiotic effects of Bacillus spp. from Pacific white shrimp (Litopenaeus vannamei) on water quality and shrimp growth, immune responses, and resistance to Vibrio parahaemolyticus (AHPND strains)[J]. Fish Shellfish Immunol, 2019, 94: 175-189. [17] CHEN C C, LI J Y, ZHANG H X, et al. Effects of a probiotic on the growth performance, intestinal flora, and immune function of chicks infected with Salmonella pullorum[J]. Poult Sci, 2020, 99(11): 5316-5323. doi: 10.1016/j.psj.2020.07.017 [18] 陈凯, 朱璐丹, 谭宏亮, 等. 2株乳酸菌抑菌作用研究及安全性评价[J]. 南方水产科学, 2019, 15(5): 118-125. doi: 10.12131/20190012 [19] FENG Z F, SONG X J, ZHAO L T, et al. Isolation of probiotics and their effects on growth, antioxidant and non-specific immunity of sea cucumber Apostichopus japonicus[J]. Fish Shellfish Immunol, 2020, 106: 1087-1094. doi: 10.1016/j.fsi.2020.08.049 [20] BENBARA T, LALOUCHE S, DRIDER D et al. Lactobacillus plantarum S27 from chicken faeces as a potential probiotic to replace antibiotics: evidence[J]. Benef Microbes, 2020, 11(2): 163-173. doi: 10.3920/BM2019.0116 [21] 布坎南 R E, 吉本斯 N E. 伯杰细菌鉴定手册[M]. 中国科学院微生物研究所《伯杰细菌鉴定手册》翻译组, 译. 北京: 科学出版社, 1984: 809. [22] YIN J Y, WANG Q, WANG Y Y, et al. Development of a simple and rapid reverse transcription-loopmediated isothermal amplification (RT-LAMP) assay for sensitive detection of tilapia lake virus[J]. J Fish Dis, 2019, 42(26): 817-824. [23] 高艳侠, 张德锋, 可小丽, 等. 罗非鱼源无乳链球菌肠道拮抗芽孢杆菌的筛选及其生物学特性[J]. 微生物学报, 2019, 59(5): 926-938. [24] 农村农业部渔业渔政管理局, 全国水产技术推广总站. 2020中国水生动物卫生状况报告[R]. 北京: 中国农业出版社, 2020: 3. [25] 蒋魁, 徐力文, 苏友禄, 等. 2012年~2014年南海海水养殖鱼类病原菌哈维弧菌分离株的耐药性分析[J]. 南方水产科学, 2016, 12(6): 99-107. doi: 10.3969/j.issn.2095-0780.2016.06.013 [26] 朱世超, 钱卓真, 吴成业. 水产品中7种大环内酯类抗生素残留量的测定HPLC-MS/MS法[J]. 南方水产科学, 2012, 8(1): 54-60. doi: 10.3969/j.issn.2095-0780.2012.01.009 [27] WANG Z, WANG L, CHEN Z, et al. In vitro evaluation of swine-derived Lactobacillus reuteri: probiotic properties and effects on intestinal porcine epithelial cells challenged with Enterotoxigenic Escherichia coli K88[J]. J Microbiol Biotechnol, 2016, 26(6): 1018-1025. doi: 10.4014/jmb.1510.10089 [28] MOHAMMADIAN T, NASIRPOUR M, TABANDEH M R, et al. Administrations of autochthonous probiotics altered juvenile rainbow trout Oncorhynchus mykiss health status, growth performance and resistance to Lactococcus garvieae, an experimental infection[J]. Fish Shellfish Immunol, 2019, 86: 269-279. doi: 10.1016/j.fsi.2018.11.052 [29] SHA Y J, WANG L, LIU M, et al. Effects of lactic acid bacteria and the corresponding supernatant on the survival, growth performance, immune response and disease resistance of Litopenaeus vannamei[J]. Aquaculture, 2016, 452: 28-36. doi: 10.1016/j.aquaculture.2015.10.014 [30] LI C, REN Y C, JIANG S H, et al. Effects of dietary supplementation of four strains of lactic acid bacteria on growth, immune-related response and genes expression of the juvenile sea cucumber Apostichopus japonicus Selenka[J]. Fish Shellfish Immunol, 2018, 74: 69-75. doi: 10.1016/j.fsi.2017.12.037 [31] De MELO P V, de OLIVEIRA C B, JÚNIOR A M, et al. How to select a probiotic? A review and update of methods and criteria[J]. Biotechnol Adv, 2018, 36(8): 2060-2076. doi: 10.1016/j.biotechadv.2018.09.003 [32] MAN L L, XIANG D J. Characterization of a broad spectrum bacteriocin produced by Lactobacillus plantarum MXG-68 from inner Mongolia traditional fermented koumiss[J]. Folia Microbiol (Praha), 2019, 64(6): 821-834. doi: 10.1007/s12223-019-00697-0 [33] 王水泉, 包艳, 董喜梅, 等. 植物乳杆菌的生理功能及应用[J]. 中国农业科技导报, 2010, 12(4): 49-55. doi: 10.3969/j.issn.1008-0864.2010.04.10 [34] 刘佳琪, 高帅, 段可馨, 等. 鱼源植物乳杆菌表达IPNV VP2-VP3重组蛋白及其口服免疫程序[J]. 水产学报, 2017, 41(4): 622-627. [35] NEWAJ F A, AL-HARBI A H, AUSTIN B. Review: developments in the use of probiotics for disease control in aquaculture[J]. Aquaculture, 2014, 431: 1-11. doi: 10.1016/j.aquaculture.2013.08.026 [36] CANDELA M, PERNA F, CARNEVALI P, et al. Interaction of probiotic Lactobacillus and Bifidobacterium strains with human intestinal epithelial cells: adhesion properties, competition against enteropathogens and modulation of IL-8 production[J]. Int J Food Microbiol, 2008, 125(3): 286-292. doi: 10.1016/j.ijfoodmicro.2008.04.012 [37] FRAQUEZA M J. Antibiotic resistance of lactic acid bacteria isolated from dry-fermented sausages[J]. Int J Food Microbiol, 2015, 212(SI): 76-88. [38] 阚刘刚, 赵丽杰, 李秀业, 等. 鸡沙门氏菌病的生物预防和控制研究进展[J]. 动物营养学报, 2018, 30(9): 3432-3443. doi: 10.3969/j.issn.1006-267x.2018.09.012 [39] 杨红玲, 孙云章, 叶继丹, 等. 2株鱼源乳酸菌的生物学特性研究[J]. 西北农林科技大学学报 (自然科学版), 2008, 36(8): 25-30. [40] 杨媛媛, 王楠楠, 曹青, 等. 鲫肠道乳酸菌的分离及益生特性[J]. 水产学报, 2018, 42(10): 1596-1605. [41] 赵芳, 李艳琴, 李彬春. 模拟人体胃肠道环境筛选益生乳杆菌[J]. 微生物学通报, 2016, 43(6): 1396-1403. [42] 龙华. 温度对鱼类生存的影响[J]. 中山大学学报 (自然科学版), 2005, 44(S1): 254-257. [43] KAKELAR H M, BARZEGARI A, HANIFIAN S, et al. Isolation and molecular identification of Lactobacillus with probiotic potential from abomasums driven rennet[J]. Food Chem, 2019, 272: 709-714. doi: 10.1016/j.foodchem.2018.08.081 [44] GHOLIZADEH P, MAHALLEI M, PORMOHAMMAD, et al. Microbial balance in the intestinal normal microbiome and its association with diabetes, obesity and allergic disease[J]. Microb Pathogenesis, 2019, 127: 48-55. doi: 10.1016/j.micpath.2018.11.031 [45] KIYMACI M E, ALTANLAR N, GUMUSTAS M A, et al. Quorum sensing signals and related virulenc inhibition of Pseudomo nasaeruginosa by a potential probiotic strain's organic acid[J]. Microb Pathogenesis, 2018, 121: 190-197. doi: 10.1016/j.micpath.2018.05.042 [46] WANG A R, RAN C, WANG Y B, et al. Use of probiotics in aquaculture of China: a review of the past decade[J]. Fish Shellfish Immunol, 2019, 86: 734-755. doi: 10.1016/j.fsi.2018.12.026 [47] CAI Y, BENNO Y, NAKASE T, et al. Specific probiotic characterization of Weissella hellenica DS-12 isolated from flounder intestine[J]. J Gen Appl Microbiol, 1998, 44(5): 311-316. doi: 10.2323/jgam.44.311 [48] WANG Y G, LEE K L, NAJIAH M, et al. A new bacterial white spot syndrome (BWSS) in cultured tiger shrimp Penaeus monodon and its comparison with white spot syndrome (WSS) caused by virus[J]. Dis Aquat Organ, 2000, 41(1): 9-18. [49] 骆艺文, 郝志凯, 王印庚, 等. 一株引起刺参“腐皮综合征”的蜡样芽胞杆菌[J]. 水产科技情报, 2009, 36(2): 60-63. doi: 10.3969/j.issn.1001-1994.2009.02.011 [50] VELMURUGAN S, PALANIKUMAR P, VELAYUTHANI P, et al. Bacterial white patch disease caused by Bacillus cereus, a new emerging disease in semi-intensive culture of Litopenaeus vannamei[J]. Aquaculture, 2015, 444(3): 49-54. [51] 闫肃, 李慧敏, 张晓冬, 等. 不同食物来源乳酸细菌的安全性评价[J]. 食品与发酵工业, 2018, 44(5): 82-89. [52] 李绍戊, 王荻, 刘红柏, 等. 鱼源嗜水气单胞菌多重耐药菌株整合子的分子特征[J]. 中国水产科学, 2013, 20(5): 1015-1022. [53] 王冉, 刘铁铮, 王恬. 抗生素在环境中的转归及其生态毒性[J]. 生态学报, 2006, 26(1): 265-270. doi: 10.3321/j.issn:1000-0933.2006.01.032 [54] 乔毅. 江苏省沿海地区水产养殖主要致病菌耐药性研究[D]. 上海: 上海海洋大学, 2015: 1-96. [55] AQUILANTI L, GAROFALO C, OSIMANI A A, et al. Isolation and molecular characterization of anti biotic-resistant lactic acid bacteria from poultry and swine meat products[J]. J Food Protect, 2007, 70(3): 557-565. doi: 10.4315/0362-028X-70.3.557 [56] ROJO-BEZARES B, SÁENZ Y, POETA P A, et al. Assessment of antibiotic susceptibility within lactic acid bacteria strains isolated from wine[J]. Int J Food Microbiol, 2006, 111(3): 234-240. doi: 10.1016/j.ijfoodmicro.2006.06.007 [57] GEVERS D, HUYS G, SWINGS J. In vitro conjugal transfer of tetracycline resistance from Lactobacillus isolates to other gram positive bacteria[J]. FEMS Microbiol Lett, 2003, 225(1): 125-130. doi: 10.1016/S0378-1097(03)00505-6 -
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