幽门螺杆菌向“存活但不可培养”状态转变的影响因素研究  

Study on the factors affecting the conversion of Helicobacter pylori to“viable but not culturable”state

作　　者：刘冬梅 潘美伶 沈丹芸 陈嘉熠 裴晓方 许欣 LIU Dong-mei;PAN Mei-ling;SHEN Dan-yun;CHEN Jia-yi;PEI Xiao-fang;XU Xin(不详;Department of Public Health Laboratory Sciences,West China School of Public Health and West China Forth Hospital,Sichuan University,Chengdu,Sichuan 610041,China)

机构地区：[1]四川大学华西公共卫生学院/华西第四医院,四川成都610041 [2]简阳市中医医院,四川简阳641400

出　　处：《现代预防医学》2022年第22期4191-4197,4202,共8页Modern Preventive Medicine

基　　金：四川省科技厅重点研发项目(2020YFS0289)。

摘　　要：目的探究幽门螺杆菌(Hp)在不同环境下转变为“存活但不可培养(VBNC)”状态的条件,为Hp的潜在传播积累资料。方法将标准菌株ATCC 43504和临床分离株Hp44、Hp65菌悬液分别用氧胁迫、热力、紫外线照射和微波处理。采用活菌计数,PMA-qPCR、LIVE/DEAD染色、革兰染色、RT-qPCR共同检测HpVBNC态生成、形态改变及毒力基因的表达。结果氧胁迫40~48 h时,Hp菌悬液中均有VBNC态菌体,由“S”型向球状转变,毒力基因的表达随着VBNC态的形成而降低。Hp菌悬液分别在56℃加热10 min,80℃加热1 min时,均有菌体以VBNC态存活,80℃加热5 min,100℃加热1 min时所有菌体被杀灭。紫外线分别照射20 min、30 min时,Hp菌悬液中均有菌体以VBNC态存活。微波处理15 s时,菌体以VBNC态存活,当处理时间≥30 s时,所有Hp菌体均被杀灭。结论Hp能够在氧胁迫、热力、紫外线照射和微波处理的条件下转变为VBNC态,其在环境中的存活情况、检出率和潜在致病性值得进一步研究。Objective To explore the conditions for the transformation of Helicobacter pylori(Hp)into“viable but not culturable”(VBNC)state under different environments,and to accumulate data for the potential transmission of Hp.Methods The bacterial suspension of standard strain ATCC43504 and the clinical isolates Hp44,Hp65 were treated with oxygen stress,heat,ultraviolet,and microwave,respectively.The surface viable bacteria count method was used to count the viable bacteria,and the PMA-qPCR and LIVE/DEAD staining were used to detect the Hp VBNC state generation.Under oxygen stress,the morphological changes of bacteria were observed by Gram staining.The expression of virulence genes of HP was detected by RT qPCR.Results After 40-48 h of oxygen stress,some of the strains survived in VBNC state.The strains changed from“S”type to spherical type,and the bacterial coloration became lighter.The expression of virulence genes decreased with the formation of VBNC state.When heated at 56℃for 10 min and 80℃for 1 min,some of the strains survived in VBNC state.When heated at 80℃for 5 min and heated at 100℃for 1 min,all the bacteria were killed.After 20 min and 30 min of UV irradiation,some of the strains survived in VBNC state.When treated with microwave for 15 s,some bacteria survived in VBNC state.When treated for more than 30 s,all Hp bacteria were killed.Conclusion Hp can transform into VBNC state under the conditions of oxygen stress,heat,ultraviolet,and microwave treatment.Its survival and potential pathogenicity in environmental samples warrant further study.

关 键 词：幽门螺杆菌 VBNC RT-QPCR PMA-qPCR LIVE/DEAD染色法 

分 类 号：R573[医药卫生—消化系统]