猪源益生乳杆菌的分离、鉴定和筛选及ERIC-PCR分型研究
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摘要
从33日龄大约克×太湖二元健康断奶仔猪消化道不同部位分离到231株疑似乳杆菌。通过耐酸性试验从其中75株菌中快速筛选出26株耐酸性好的菌株：ZN8、SJBZ4、JJN1、SJNH7、SJBH4、ZB5、MNZ8、SNZ10、SJNZ4、WN8、WB5、HN1、JJB5、MBZ3、SBZ5、SBQ9、SNQ5、MNQ4、KBQ4、WN1、WB10、JJB3、ZB3、MBH5、KNZ4。
通过运动性试验、硝酸盐还原试验、明胶液化试验、吲哚(靛基质)试验、硫化氢试验和过氧化氢酶试验将26株菌鉴定为乳杆菌属。碳水化合物发酵产酸试验把26株菌鉴定为5种乳杆菌：食淀粉乳杆菌(Lactobacillus amylovorus)7株、唾液乳杆菌(Lactobacillus salivarius)3株、罗伊氏乳杆菌(Lactobacillus reuteri)6株、植物乳杆菌(Lactobacillus plantarum)9株、嗜酸乳杆菌(Lactobacillus acidophilus)1株。而根据16S rRNA基因测序结果鉴定为3种乳杆菌，即罗伊氏乳杆菌(L．reuteri)10株、约氏乳杆菌(Lactobacillus johnsonii)15株、类肠膜魏斯氏菌(Weissella paremesenteroides)1株。
采用牛津杯法进行抑菌试验，从26株菌中筛选出对大肠杆菌O_8(Escherichia coli O_8)和沙门氏菌339(Salmonella 339)均有较强抑制作用的6株乳杆菌：ZB3、WN8、WN1、SNQ5、SBZ5、JJB3。对这6株乳杆菌进一步做耐胆盐试验、耐热性试验、生长试验，最终筛选出各种性能都很优良的2株乳杆菌：SBZ5和JJB3。用利福平MRS平板(利福平含量：10 mg／L)将这2株乳杆菌驯化成耐利福平的菌株，用于28日龄断奶仔猪的饲喂试验。
“肠道细菌基因间重复序列”(Enterobacterial Repetitive Intergenic Consensus，ERIC)，也称为“基因间重复单位”(Intergenic Repetitive Unit，IRU)，是一类广泛存在于肠杆菌科细菌的重复序列。由于其在不同细菌种属甚至同一种内不同菌株之间的拷贝数和定位都不同，所以可结合PCR技术用于细菌的分类、鉴定与分型。
本实验用于乳杆菌的ERIC-PCR反应体系：总体积20μL，各成分工作浓度如下：Tris-HCl 10 mmol／L，KCl 50 mmol／L，MgCl_2 3 mmol／L，dNTP 250μmol／L，ERIC1R(5′-ATGTAAGCTCCTGGGGATTCAC-3′)和ERIC2(5′-AAGTAAGTGACTGGGGTGAGCG-3′)均为1μmol／L，Long Taq DNA聚合酶0.05 U，模板DNA 1μL。反应程序：预变性：94℃，5 min；循环反应分为前后两个部分：前5个循环：变性：94℃，1 min；退火：32℃，1 min；延伸：72℃，2 min；后40个循环：变性：94℃，
0.5 min；退火：48℃，0.5 min；延伸：72℃，1 min；最后延伸：72℃，10 min。
从本试验基于ERIC-PCR构建的菌株的聚类图来看，ERIC-PCR用于乳杆菌分型的分辨力可达到菌株水平。前述的26株菌的ERIC-PCR条带大小在200 bp-2000 bp之间，聚类分析可把26株菌分为明确的三个类群：即26-A：7株罗伊氏乳杆菌(L.reuteri)(ZN8、SJBZ4、JJN1、SJNH7、SJBH4、ZB5、MNZ8)；26-B：15株约氏乳杆菌(L.johnsonii)(SNZ10、SJNZ4、WN8、WB5、HN1、JJB5、MBZ3、SBZ5、SBQ9、SNQ5、MNQ4、KBQ4、WN1、WB10、JJB3)；26-C：3株罗伊氏乳杆菌(L.reuteri)(ZB3、MBH5、KNZ4)。26-A的各菌株图谱相似率范围为52％-76％，26-B的各菌株图谱相似率范围为40％-92％，26-C的各菌株图谱相似率范围为37％-73％。如果2株乳杆菌的图谱相似率达到75％就可判定为同一菌株，则26-A的ZN8和SJBZ4为同一菌株，SJNH7和SJBH4为同一菌株；26-B的WN8、WB5、HNl和JJB5为同一菌株，SBZ5、SBQ9和SNQ5为同一菌株；26-C的3株菌则彼此都不相同。
231 suspected Lactobacillus strains were isolated from gastrointestinal tract of Yorkshire×Taihu weaning piglet. 26 bacteria strains (ZN8、SJBZ4、JJN1、SJNH7、SJBH4、ZB5、MNZ8、SNZ10、SJNZ4、WN8、WB5、HN1、JJB5、MBZ3、SBZ5、SBQ9、SNQ5、MNQ4、KBQ4、WN1、WB10、JJB3、ZB3、MBH5、KNZ4) were screened by acid resistance test from 75 bacteria strains.
The 26 bacteria strains were identified as the members of the genus Lactobacillus by mobility test, nitrate reduction test, gelatin liquefaction test, indole test, hydrogen sulfide test and catalase test. 5 Lactobacillus species were identified by carbohydrate fermentation test: 7 Lactobacillus amylovorus strains, 3 Lactobacillus salivarius strains, 6 Lactobacillus reuteri strains, 9 Lactobacillus plantarum strains, 1 Lactobacillus acidophilus strains. Meanwhile, 3 Lactobacillus species were identified by 16S rRNA gene sequencing: 10 L. reuteri strains, 15 Lactobacillus johnsonii strains, 1 Weissella paremesenteroides strain.
6 Lactobacillus strains, ZB3, WN8, WN1, SNQ5, SBZ5 and JJB3, which have strong inhibition on both Escherichia coli O_8 and Salmonella 339 were screened from the 26 bacteria strains by bacteriostatic test. 2 best Lactobacillus strains, SBZ5 and JJB3, were screened by bile tolerance test, heat-resistance test and growth test. Spontaneous rifampin-resistant (Rif~1) variants of the 2 Lactobacillus strains were isolated by spread plating 10~9 CFU from an overnight culture onto MRS agar containing 10mg of rifampm/L and then were applied in weaning piglet feeding.
Enterobacterial Repetitive Intergenic Consensus (ERIC), also named Intergenic Repetitive Unit (IRU), is a kind of intergenic repetitive sequence that exists predo minantly in Enterobacteria. ERIC sequences are dispersed throughout bacterial genomes and their copies or inter-ERIC distances are typical for given bacterial species and sometimes even for strains within a given species. So it can be used for classification, identification and differentiation of bacteria.
The 20μL reaction mixture contained 10 mmol/L Tris-HCl, 50 mmol/L KC1, 3 mmol/L MgCl_2, 250μmol/L of each deoxynucleoside triphosphate, 1μmol/L of each primer (ERIC1R, 5'-ATGTAAGCTCCTGGGGATTCAC-3' and ERIC2, 5'-AAGTAAGTGACTG GGGTGAGCG-3'), 0.05 U of Long Taq DNA polymerase and 1μL of the respective template DNA. Samples were denatured for 5 min at 94℃and processed in the following manner: one cycle at 94℃for 3 min; followed by 5 cycles at 94℃for 1 min, at 32℃for 1 min, at 72℃for 2 min and 40 cycles at 94℃for 0.5 min, at 48℃for 0.5 min, at 72℃for 1 min and one cycle at 72℃for 7 min.
ERIC-PCR was confirmed to be a powerful tool for a very rapid and highly reliable differentiation of Lactobacillus isolates at their strain level. ERIC-PCR for the 26 bacteria strains generated multiple DNA fragments ranging from 200 bp to 2000 bp with various intensities. The 26 isolates were grouped into 3 different groups with clearly distinguishable ERIC-PCR patterns, named as 26-A, 26-B, 26-C. The cluster 26-A included 7 L. reuteri strains(ZN8、SJBZ4、JJN1、SJNH7、SJBH4、ZB5、MNZ8), while 26-B and 26-C harbored 15 L. johnsonii strains (SNZ10、SJNZ4、WN8、WB5、HN1、JJB5、MBZ3、SBZ5、SBQ9、SNQ5、MNQ4、KBQ4、WN1、WB10、JJB3)and 3 L. reuteri strains (ZB3、MBH5、KNZ4) respectively. The genetic similarity value of group 26-A ranged between 52%-76%, while that of 26-B and 26-C ranged between 40%-92% and 37%-73% respectively. If 75% was set as the minimum genetic similarity value of identifying two strains as the same, 2 strains (ZN8 and SJBZ4) and 2 strains (SJNH7 and SJBH4) out of 26-A were the same strain, while 4 strains (WN8, WB5, HN1 and JJB5) and 3 stains (SBZ5, SBQ9 and SNQ5) out of 26-B were the same strain respectively. In addition, the 3 strains of 26-C were different from each other.
通过运动性试验、硝酸盐还原试验、明胶液化试验、吲哚(靛基质)试验、硫化氢试验和过氧化氢酶试验将26株菌鉴定为乳杆菌属。碳水化合物发酵产酸试验把26株菌鉴定为5种乳杆菌：食淀粉乳杆菌(Lactobacillus amylovorus)7株、唾液乳杆菌(Lactobacillus salivarius)3株、罗伊氏乳杆菌(Lactobacillus reuteri)6株、植物乳杆菌(Lactobacillus plantarum)9株、嗜酸乳杆菌(Lactobacillus acidophilus)1株。而根据16S rRNA基因测序结果鉴定为3种乳杆菌，即罗伊氏乳杆菌(L．reuteri)10株、约氏乳杆菌(Lactobacillus johnsonii)15株、类肠膜魏斯氏菌(Weissella paremesenteroides)1株。
采用牛津杯法进行抑菌试验，从26株菌中筛选出对大肠杆菌O_8(Escherichia coli O_8)和沙门氏菌339(Salmonella 339)均有较强抑制作用的6株乳杆菌：ZB3、WN8、WN1、SNQ5、SBZ5、JJB3。对这6株乳杆菌进一步做耐胆盐试验、耐热性试验、生长试验，最终筛选出各种性能都很优良的2株乳杆菌：SBZ5和JJB3。用利福平MRS平板(利福平含量：10 mg／L)将这2株乳杆菌驯化成耐利福平的菌株，用于28日龄断奶仔猪的饲喂试验。
“肠道细菌基因间重复序列”(Enterobacterial Repetitive Intergenic Consensus，ERIC)，也称为“基因间重复单位”(Intergenic Repetitive Unit，IRU)，是一类广泛存在于肠杆菌科细菌的重复序列。由于其在不同细菌种属甚至同一种内不同菌株之间的拷贝数和定位都不同，所以可结合PCR技术用于细菌的分类、鉴定与分型。
本实验用于乳杆菌的ERIC-PCR反应体系：总体积20μL，各成分工作浓度如下：Tris-HCl 10 mmol／L，KCl 50 mmol／L，MgCl_2 3 mmol／L，dNTP 250μmol／L，ERIC1R(5′-ATGTAAGCTCCTGGGGATTCAC-3′)和ERIC2(5′-AAGTAAGTGACTGGGGTGAGCG-3′)均为1μmol／L，Long Taq DNA聚合酶0.05 U，模板DNA 1μL。反应程序：预变性：94℃，5 min；循环反应分为前后两个部分：前5个循环：变性：94℃，1 min；退火：32℃，1 min；延伸：72℃，2 min；后40个循环：变性：94℃，
0.5 min；退火：48℃，0.5 min；延伸：72℃，1 min；最后延伸：72℃，10 min。
从本试验基于ERIC-PCR构建的菌株的聚类图来看，ERIC-PCR用于乳杆菌分型的分辨力可达到菌株水平。前述的26株菌的ERIC-PCR条带大小在200 bp-2000 bp之间，聚类分析可把26株菌分为明确的三个类群：即26-A：7株罗伊氏乳杆菌(L.reuteri)(ZN8、SJBZ4、JJN1、SJNH7、SJBH4、ZB5、MNZ8)；26-B：15株约氏乳杆菌(L.johnsonii)(SNZ10、SJNZ4、WN8、WB5、HN1、JJB5、MBZ3、SBZ5、SBQ9、SNQ5、MNQ4、KBQ4、WN1、WB10、JJB3)；26-C：3株罗伊氏乳杆菌(L.reuteri)(ZB3、MBH5、KNZ4)。26-A的各菌株图谱相似率范围为52％-76％，26-B的各菌株图谱相似率范围为40％-92％，26-C的各菌株图谱相似率范围为37％-73％。如果2株乳杆菌的图谱相似率达到75％就可判定为同一菌株，则26-A的ZN8和SJBZ4为同一菌株，SJNH7和SJBH4为同一菌株；26-B的WN8、WB5、HNl和JJB5为同一菌株，SBZ5、SBQ9和SNQ5为同一菌株；26-C的3株菌则彼此都不相同。
231 suspected Lactobacillus strains were isolated from gastrointestinal tract of Yorkshire×Taihu weaning piglet. 26 bacteria strains (ZN8、SJBZ4、JJN1、SJNH7、SJBH4、ZB5、MNZ8、SNZ10、SJNZ4、WN8、WB5、HN1、JJB5、MBZ3、SBZ5、SBQ9、SNQ5、MNQ4、KBQ4、WN1、WB10、JJB3、ZB3、MBH5、KNZ4) were screened by acid resistance test from 75 bacteria strains.
The 26 bacteria strains were identified as the members of the genus Lactobacillus by mobility test, nitrate reduction test, gelatin liquefaction test, indole test, hydrogen sulfide test and catalase test. 5 Lactobacillus species were identified by carbohydrate fermentation test: 7 Lactobacillus amylovorus strains, 3 Lactobacillus salivarius strains, 6 Lactobacillus reuteri strains, 9 Lactobacillus plantarum strains, 1 Lactobacillus acidophilus strains. Meanwhile, 3 Lactobacillus species were identified by 16S rRNA gene sequencing: 10 L. reuteri strains, 15 Lactobacillus johnsonii strains, 1 Weissella paremesenteroides strain.
6 Lactobacillus strains, ZB3, WN8, WN1, SNQ5, SBZ5 and JJB3, which have strong inhibition on both Escherichia coli O_8 and Salmonella 339 were screened from the 26 bacteria strains by bacteriostatic test. 2 best Lactobacillus strains, SBZ5 and JJB3, were screened by bile tolerance test, heat-resistance test and growth test. Spontaneous rifampin-resistant (Rif~1) variants of the 2 Lactobacillus strains were isolated by spread plating 10~9 CFU from an overnight culture onto MRS agar containing 10mg of rifampm/L and then were applied in weaning piglet feeding.
Enterobacterial Repetitive Intergenic Consensus (ERIC), also named Intergenic Repetitive Unit (IRU), is a kind of intergenic repetitive sequence that exists predo minantly in Enterobacteria. ERIC sequences are dispersed throughout bacterial genomes and their copies or inter-ERIC distances are typical for given bacterial species and sometimes even for strains within a given species. So it can be used for classification, identification and differentiation of bacteria.
The 20μL reaction mixture contained 10 mmol/L Tris-HCl, 50 mmol/L KC1, 3 mmol/L MgCl_2, 250μmol/L of each deoxynucleoside triphosphate, 1μmol/L of each primer (ERIC1R, 5'-ATGTAAGCTCCTGGGGATTCAC-3' and ERIC2, 5'-AAGTAAGTGACTG GGGTGAGCG-3'), 0.05 U of Long Taq DNA polymerase and 1μL of the respective template DNA. Samples were denatured for 5 min at 94℃and processed in the following manner: one cycle at 94℃for 3 min; followed by 5 cycles at 94℃for 1 min, at 32℃for 1 min, at 72℃for 2 min and 40 cycles at 94℃for 0.5 min, at 48℃for 0.5 min, at 72℃for 1 min and one cycle at 72℃for 7 min.
ERIC-PCR was confirmed to be a powerful tool for a very rapid and highly reliable differentiation of Lactobacillus isolates at their strain level. ERIC-PCR for the 26 bacteria strains generated multiple DNA fragments ranging from 200 bp to 2000 bp with various intensities. The 26 isolates were grouped into 3 different groups with clearly distinguishable ERIC-PCR patterns, named as 26-A, 26-B, 26-C. The cluster 26-A included 7 L. reuteri strains(ZN8、SJBZ4、JJN1、SJNH7、SJBH4、ZB5、MNZ8), while 26-B and 26-C harbored 15 L. johnsonii strains (SNZ10、SJNZ4、WN8、WB5、HN1、JJB5、MBZ3、SBZ5、SBQ9、SNQ5、MNQ4、KBQ4、WN1、WB10、JJB3)and 3 L. reuteri strains (ZB3、MBH5、KNZ4) respectively. The genetic similarity value of group 26-A ranged between 52%-76%, while that of 26-B and 26-C ranged between 40%-92% and 37%-73% respectively. If 75% was set as the minimum genetic similarity value of identifying two strains as the same, 2 strains (ZN8 and SJBZ4) and 2 strains (SJNH7 and SJBH4) out of 26-A were the same strain, while 4 strains (WN8, WB5, HN1 and JJB5) and 3 stains (SBZ5, SBQ9 and SNQ5) out of 26-B were the same strain respectively. In addition, the 3 strains of 26-C were different from each other.
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