四川泡菜中乳酸菌链霉素抗性与抗性基因的检测
目的 探明四川泡菜中链霉素抗性乳酸菌及抗性基因的种类。方法 利用含有8 μg/ml链霉素的MRS初步分离泡菜液中的抗性乳酸菌，通过16S rRNA分析确定抗性乳酸菌的分类地位后，测定同种不同菌株对链霉素的MIC，并与欧洲食品安全官方机构（EFSA）建议的最低临界值比较确定抗性菌株；通过PCR扩增链霉素抗性基因strA、strB, aadA、aadE、ant(6)、aac(6')-aph(2')和aph(3')-Ⅲa，确定抗性菌株的抗性基因。结果 分离到67株链霉素敏感性或抗性菌株，这些菌株分别属于Pediococcus ethanolidurans (36), Lactococcus garvieae (14), Lactobacillus buchneri (12), Lactobacillus acetotolerans (2), Lactococcus lactis (1)和Staphylococcus.spp (2)。其中Lactococcus garvieae、Lactobacillus acetotolerans、Lactococcus lactis和Staphylococcus.spp全部为抗性菌株，Pediococcus ethanolidurans中有抗性菌株20株、Lactobacillus buchneri 中有7株。在抗性基因检测中，除Lactobacillus acetotolerans抗性菌株没有检测到被检基因外，其他5个种的抗性菌株中均检测到部分或全部抗性基因。strA 和aph(3')-Ⅲa基因在除Lactobacillus acetotolerans外的被检菌株中均有检出，其检出率分别为50%-100%和21.4%-100%；strB基因在抗性菌株Pediococcus ethanolidurans, Lactobacillus buchneri, Lactococcus garvieae和Lactococcus lactis检测率分别为70%、42.9%、28.6%和100%；aac(6')-aph(2')基因仅在3株Pediococcus ethanolidurans、1株Lactobacillus buchneri、1株Lactococcus garvieae和Staphylococcus spp中检测到。aadA、aadE and ant (6)在所有抗性菌株中都没有检测到。总体而言，strA、strB和aph(3')-Ⅲa基因在链霉素抗性菌株中的检出率高于其他抗性基因。结论 当前的研究结果表明：四川泡菜中存在链霉素乳酸菌抗性菌株，这些抗性菌株对四川泡菜存在潜在安全风险。
Objective To detect the streptomycin resistance and resistance genes of lactic acid bacteria (LAB) in Sichuan Pickle of China. Methods The streptomycin resistant LABs were isolated by MRS containing 8 μg/ml streptomycin, and were identified by 16S rRNA analysis. The minimum inhibitory concentration (MIC) for streptomycin was determined for each isolate by broth microdilution method. And then suscep-tibility status was determined by comparing MIC values to breakpoints proposed by European Food Safety Authority (EFSA). In the streptomycin resistant LABs, the candidate streptomycin resistance genes (strA, strB, aadA, aadE, ant(6), aac(6')-aph(2'), and aph(3')- Ⅲa) were detected by PCR. Results Sixty-seven lactic acid bacteria which belonged to Pediococcus ethanolidurans (36), Lactococcus garvieae (14), Lactobacillus buchneri (12), Lactobacillus acetotolerans (2), Lactococcus lactis (1) and Staphylococcus.spp (2) were isolated from Sichuan Pickle of China. In these isolates, all Lactococcus garvieae, Lactobacillus acetotolerans, Lactococcus lactis and Staphylococcus.spp strains displayed streptomycin resistance, while only 20 Pediococcus ethanolidurans and 7 Lactobacillus buchneri isolates were streptomycin resistant. Except Lactobacillus acetotolerans, isolates belonged to the other 5 species harbored several or all candidate resistance genes. The strA and aph(3')-Ⅲa genes were detected in all 5 species isolates with detection rate of 50% - 100% and 21.4% - 100%, respectively. The detection rates of strB gene in Pediococcus ethanolidurans, Lactobacillus buchneri, Lactococcus garvieae and Lactococcus lactis were 70%, 42.9%, 28.6% and 100%, respectively. The aac(6') - aph(2') genes were detected only in 3 Pediococcus ethanolidurans, one Lactobacillus buchneri, one Lactococcus garvieae and 2 Staphylococcus spp strains. And no amplicon resistance genes such as aadA, aadE and ant(6) were detected in either isolate. In conclusion, the detection rates of strA, strB and aph(3')-Ⅲa genes were more than other genes. Conclusion These results indicated that lactic acid bacteria with streptomycin resistance were living in the brine of Sichuan Pickle, and it would be a potential health risk.
标题：四川泡菜中乳酸菌链霉素抗性与抗性基因的检测
英文标题：Detection of streptomycin resistance and resistance genes in lactic acid bacteria from Sichuan Pickle of China
作者：
宋菲菲 西华大学生物工程学院,四川省食品生物技术重点实验室,西华大学古法发酵(酿造)生物技术研究所
徐顾榕 西华大学生物工程学院,四川省食品生物技术重点实验室,西华大学古法发酵(酿造)生物技术研究所
蔡婷 西华大学生物工程学院,四川省食品生物技术重点实验室,西华大学古法发酵(酿造)生物技术研究所
林凯 西华大学生物工程学院,四川省食品生物技术重点实验室,西华大学古法发酵(酿造)生物技术研究所
贾碧洪 西华大学生物工程学院,四川省食品生物技术重点实验室,西华大学古法发酵(酿造)生物技术研究所
陈功 四川省食品发酵工业研究设计院
向文良 西华大学生物工程学院,四川省食品生物技术重点实验室,西华大学古法发酵(酿造)生物技术研究所
张庆 西华大学生物工程学院,四川省食品生物技术重点实验室,西华大学古法发酵(酿造)生物技术研究所
中文关键词:四川泡菜,乳酸菌,链霉素抗性,链霉素抗性基因,
英文关键词:Sichuan Pickle,lactic acid bacteria,streptomycin resistance,streptomycin resistance genes,
发表日期：2014-11-11
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