Isolation and identification of culturable intestinal bacteria from Clogmia albipunctata larvae and analysis of their digestive enzyme-producing activities

doi:10.11853/j.issn.1003.8280.2021.02.021

Abstract

Abstract: Objective To isolate culturable intestinal bacteria from Clogmia albipunctata larvae, determine the digestive enzyme-producing activity and drug resistance of the bacteria, and to explore the effects of intestinal bacteria on food digestion and acclimatization of C. albipunctata. Methods The traditional isolation and culture method was used for the isolation and purification of culturable intestinal bacteria from C. albipunctata larvae, and 16S rRNA sequencing was used for identification. The flat transparent circle method was applied to screen for bacteria producing amylase, protease, cellulase, or lipase. The ratio of transparent circle diameter (D) to bacterial colony diameter (d) was calculated to compare the digestive enzyme-producing activities of different bacteria. Statistical analysis was performed using SPSS 20.0 software. The one-way analysis of variance was used for comparison between groups. When the variance is homogeneous, S-N-K multiple comparison was performed; when the variance is not homogeneous, the Dunnett's T3 test was performed. The paper disk diffusion method was used to analyze the drug susceptibility of different culturable intestinal bacteria. Results A total of 12 species of bacteria belonging to 5 genera were isolated from the intestine of C. albipunctata larvae. There were 7 species belonging to the genus Bacillus-B. vietnamensis, B. subtilis subsp., B. thuringiensis, B. aryabhattai, B. cereus, Lysinibacillus fusiformis, and L. pakistanensis. There were 2 species belonging to the genus Enterococcus-E. casseliflavus and E. gallinarum. There was 1 species each belonging to the genus Exiguobacterium, Leucobacter, and Staphylococcus-E. profundum, L. celer subsp., and S. sciuri. Among these bacteria, 8 species showed amylase-producing activity, 6 species showed protease-producing activity, and no bacteria showed cellulase- or lipase-producing activity. There was a significant difference in amylase-producing activity between the 8 species of bacteria (F=8.779, P<0.001). B. vietnamensis, B. subtilis subsp. and E. profundum had relatively high activity of producing amylase. There was a significant difference in protease-producing activity between the 6 species of bacteria (F=23.980, P<0.001). B. vietnamensis and B. subtilis subsp. had relatively high activity of producing protease. In drug susceptibility tests, S. sciuri showed resistance to 6 antibiotics, E. gallinarum were resistant to 5 antibiotics, and B. aryabhattai and E. casseliflavus showed resistance to 4 antibiotics. Conclusion The culturable intestinal bacteria from C. albipunctata larvae have the ability to produce amylase and protease, which may assist in digestion and breakdown of food. The intestinal bacteria generally have certain drug resistance, which can help C. albipunctata larvae survive in a dirty environment.

Key words: Clogmia albipunctata larvae, Intestinal bacteria, Digestive enzyme-producing bacteria, Drug resistance

摘要： 目的分离白斑蛾蚋幼虫肠道可培养细菌，并测定其产消化酶活性和耐药性，探讨肠道细菌对白斑蛾蚋幼虫消化食物及其环境适应的影响。方法采用传统分离培养的方法，分离纯化白斑蛾蚋幼虫肠道可培养细菌，通过16S rRNA序列分析进行分子鉴定，利用平板透明圈法筛选产淀粉酶、蛋白酶、纤维素酶和脂肪酶的细菌，通过水解圈（D）与菌落直径（d）的比值，比较不同细菌的产消化酶活性。利用SPSS 20.0软件进行统计学分析，组间比较采用单因素方差分析，当方差齐时用SNK多重比较方法；方差不齐时用Dunnett's T3进行显著性分析。针对不同肠道可培养细菌采用纸片扩散法进行药敏试验分析。结果在白斑蛾蚋幼虫肠道中分离到可培养细菌有5属12种细菌，其中芽孢杆菌属7种，分别为越南芽孢杆菌、枯草芽孢杆菌枯草亚种、苏云金芽孢杆菌、阿氏芽孢杆菌、蜡状芽孢杆菌、纺锤形赖氨酸芽孢杆菌和巴基斯坦短小芽孢杆菌；肠球菌属2种，分别为铅黄肠球菌、鹑鸡肠球菌；微小杆菌属、白色杆菌属和葡萄球菌属各1种，分别为深海微小杆菌，Leucobacter celer subsp.和松鼠葡萄球菌。在分离到的白斑蛾蚋肠道可培养细菌中，可产淀粉酶活性的细菌有8种，产蛋白酶活性的细菌有6种，未筛选到产纤维素酶和脂肪酶活性的细菌。8种细菌的产淀粉酶活性差异有统计学意义（F=8.779，P<0.001）。其中越南芽孢杆菌、枯草芽孢杆菌枯草亚种和深海微小杆菌的产淀粉酶活性较强；6种细菌的产蛋白酶活性差异有统计学意义（F=23.980，P<0.001），其中越南芽孢杆菌和枯草芽孢杆菌枯草亚种产蛋白酶活性较强。药敏实验表明，松鼠葡萄球菌对6种抗生素耐药，鹑鸡肠球菌对5种抗生素耐药，阿氏芽孢杆菌和铅黄肠球菌对4种抗生素耐药。结论白斑蛾蚋幼虫肠道可培养细菌具有产淀粉酶和蛋白酶能力，可能协助消化和分解食物。肠道细菌普遍具有一定的耐药性，有助于白斑蛾蚋幼虫在污浊的环境中更好的生存。

关键词: 白斑蛾蚋幼虫, 肠道细菌, 产消化酶细菌, 耐药性

CLC Number:

R384.5

HE Zhi-wei, DONG Qi-yu, LIU Rui, LI Xin, LIU Xin, XU Cong-wu, ZHAO Xin-qi, WAN Qing, ZHANG Zhong. Isolation and identification of culturable intestinal bacteria from Clogmia albipunctata larvae and analysis of their digestive enzyme-producing activities[J]. Chines Journal of Vector Biology and Control, 2021, 32(2): 224-229.

何志伟, 董起钰, 刘锐, 李欣, 刘欣, 徐从武, 赵鑫祺, 万晴, 张忠. 白斑蛾蚋幼虫肠道可培养细菌的分离鉴定与产消化酶活性分析[J]. 中国媒介生物学及控制杂志, 2021, 32(2): 224-229.

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