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摘要：【目的】筛选出具有高效产酶活性和一定耐药性的芽孢杆菌，为开发本土高效的水产养殖益生菌提供参考依据。【方法】从广东湛江市海滨公园红树林采集淤泥样品，通过菌株富集和功能筛选，采用形态特征和生理生化特征、16S rDNA序列及gyrB基因克隆等方法对分离菌株进行鉴定，并开展几种常用渔药（复方新诺明、甲氧苄胺嘧啶、氨苄西林和四环素）的药敏试验。【结果】从广东湛江市海滨公园红树林淤泥中分离获得两株生长速度快且功能较强的菌株，分别命名为YB-3和YB-5。菌株YB-3的氨氮降解和淀粉水解能力较强，可在7% NaCl、9% NaCl、4 ℃、50 ℃、pH 5.7、pH 9.5的条件下生长，其16S rDNA序列长度1459 bp、gyrB基因长度1178 bp，综合形态学、生理生化及系统发育进化树分析，确定菌株YB-3為弯曲芽孢杆菌（Bacillus flexus）。菌株YB-5的酪蛋白降解能力较强，可在7% NaCl、4 ℃、50 ℃、60 ℃、pH 5.7、pH 9.5的条件下生长，其16S rDNA序列长度1461 bp、gyrB基因长度1181 bp，综合形态学、生理生化及系统发育进化树分析，确定菌株YB-5为蜡样芽孢杆菌（B. cereus）。药敏试验结果显示，菌株YB-3对四环素、氨苄西林和利福平敏感，对甲氧苄胺嘧啶和复方新诺明不敏感（耐药）;菌株YB-5对四环素敏感，对氨苄西林、甲氧苄胺嘧啶、复方新诺明和利福平不敏感（耐药）。【结论】从广东湛江市海滨公园红树林淤泥中分离获得两株芽孢杆菌，其中一株为弯曲芽孢杆菌，具有较强的氨氮降解和淀粉水解能力;另一株为蜡样芽孢杆菌，具有较强的酪蛋白降解能力。两株芽孢杆菌均具有一定的耐低温、高温特性和耐盐、耐酸碱等特性，且具有耐复方新诺明和甲氧苄胺嘧啶特性，可进一步发掘其在饲料加工、促营养物质吸收利用及降解腐殖质、缓解环境污染等方面的应用价值，或与其他具有益生功能的菌株制成复合型菌剂。
关键词： 芽孢杆菌;功能筛选;鉴定;产酶活性;抗逆特性;耐药性
中图分类号： S963.739 文献标志码： A 文章编号：2095-1191（2018）12-2551-08
Screening identification and drug sensitivity test of two marine silt Bacillus strains
CHEN Rong， ZHONG Min-juan， LIU Hong-e， SU Kai， WANG Li-yun，
CHEN Bao-ting， XIE Hai-qi， ZHANG Mei-ling， RAO Ying-zhu*
（Institute of Applied Biotechnology， Lingnan Normal University/School of Life Science and Technology， Lingnan Normal University，Zhanjiang， Guangdong 524048， China）
Abstract：【Objective】Bacillus strains with high-efficient enzyme producing activity and drug resistance were screened to provide reference for developing local high-efficient aquaculture probiotics. 【Method】Marine silt samples in Seaside Park of Zhanjiang， Guangdong were collected for strains eichment and function screening. The isolated strains were identified by morphological observation，physiological and biochemical characters， analysis of sequence of 16S rDNA and gyrB gene cloning. The drug resistance test was carried out on the strains for some common fishery drugs（compound sulfamethoxazole， trimethoprim， ampicillin and tetracycline）. 【Result】Two strains named YB-3 and YB-5 with rapid growth and powerful functions were isolated from marine mangrove silt samples from Seaside Park of Zhanjiang， Guangdong. The strain YB-3 had strong ability to degrade ammonia nitrogen and starch hydrolysis， and survived under the condition of 7% NaCl， 9% NaCl， 4 ℃， 50 ℃， pH 5.7 and pH 9.5. The length of 16S rDNA sequence of strain YB-3 was 1459 bp and the length of gyrB gene was 1178 bp. The strain YB-5 had certain activity of casein degradation， and survived under the condition of 7% NaCl， 4 ℃， 50 ℃， 60 ℃， pH 5.7 and pH 9.5. The length of 16S rDNA sequence and gyrB gene were 1461 and 1181 bp respectively. The results of identification by morphological observation， physiological and biochemical tests and phylogenetic analysis indicated that the strain YB-3 was B. flexus and the strain YB-5 was B. cereus. The drug sensitivity test results showed that the strain YB-3 was sensitive to tetracycline， ampicillin， rifampin， and was not sensitive to trimethoprim and compound sulfamethoxazole（drug resistant）， while the strain YB-5 was sensitive to tetracycline and not sensitive to ampicillin，trimethoprim，compound sulfamethoxazole and rifampin（drug resistant）. 【Conclusion】Two strains of Bacillus are isolated from silt in Seaside Park of Zhanjiang， Guangdong， one strain of strong ability of ammonia nitrogen degradation and starch hydrolysis is B. flexus and another strain is B. cereus， which has the strong ability of degradation of casein. Two strains of Bacillus are of low temperature resistant， high temperature properties and resistance to salt， acid and alkali， and both strains are resistant to compound sulfamethoxazole and trimethoprim.The application value of the two strains can be further explored for the feed processing， promoting nutrient uptake and degradation of humus， alleviating environment pollution， and making compound inoculants with other probiotics.
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