单增李斯特菌inl A/inl B双基因缺失株生长特性观察及对小鼠致病性研究

Biological characters and pathogenicity of Listeria monocytogenes △inlA/△inlB double gene deletion strains

目的探究单增李斯特菌inl A/inl B双基因缺失株的生物学特性。方法通过体外胁迫培养条件下OD600测定以及小鼠感染试验,对Lm90-△inlAB和Lm90的抗胁迫能力及细菌致病力进行研究。结果低温(4℃、30℃)环境下,Lm90和Lm90-△inlAB生长差异无统计学意义(t4℃=0.057,P>0.05;t30℃=0.441,P>0.05),适温37℃及42℃高温环境下,Lm90-△inlAB较Lm90生长受到抑制,生长差异具有统计学意义(t37℃=0.763,P<0.05;t42℃=1.147,P<0.05);体外耐酸碱试验中,Lm90-△inlAB耐酸性未明显改变,而耐碱能力强于亲本株Lm90,生长差异具有统计学意义(pH=9时t=2.837,P<0.05);在含有3.5%酒精的BHI培养基及含5%NaCl高渗BHI培养基中,Lm90-△inlAB增长趋势明显低于Lm90,生长差异具有统计学意义(t酒精=1.422,P<0.05;tNaCl=1.382,P<0.05),Lm90-△inlAB对酒精及高盐的耐受力显著低于Lm90;小鼠毒力测定结果表明,Lm90-△inlAB与Lm90半数致死量分别为106.94 CFU、105.68 CFU,Lm90-△inlAB毒力下降明显,在不同检测时间点Lm90-△inlAB在小鼠肝脏和脾脏内的载菌量均少于Lm90,载菌量差异具有统计学意义(t肝=2.454,P<0.05;t脾=2.443,P<0.05)。结论 Lm的抗胁迫能力可能随着基因的缺失发生显著改变,内化素InlA/InlB与单增李斯特菌侵染宿主的能力有一定的调控作用。缺失株生物学特性测定对研究Lm胁迫环境生存及毒力因子的致病机理提供了科学依据。

In order to explore the biological characters of the△inlA/△inlB double gene deletion strains of Listeria monocytogenes,the ability to resist stress and pathogenicity of Lm90 and Lm90-△inlAB were studied by in vitro stress culture and mouse infection experiment.Results showed that there was no significant difference（t4℃ =0.057,P〉0.05;t30℃ =0.441,P〉0.05）between Lm90 and Lm90-△inlAB at low temperature（4-30℃）and the growth of Lm90 was better than that of Lm90-△inlAB at 37 ℃ and 42 ℃（t37 ℃=0.763,P〈0.05;t42 ℃=1.147,P〈0.05）.For acid and alkali resistance test in vitro,there was no obvious change of acid resistant ability but a better alkali resistant ability of Lm90-△inlAB than parent strain Lm90（pH=9,t=2.837,P〈0.05）.The growth of Lm90-△inlAB was significantly lower than Lm90 in BHI culture medium containing 3.5% alcohol and 5% NaCl hypertonic BHI medium and the difference was significant（talcohol=1.422,P〈0.05;tNaCl=1.382,P〈0.05）.In other words,the tolerance of alcohol and high salt of Lm90-△inlAB was significantly lower than that of Lm90.Results of mice virulence determination showed that the median lethal dose of Lm 90-△inlAB and Lm 90 were 106.94 CFU and105.68 CFU respectively,and the virulence of Lm90-△inlAB decreased significantly.The microbial load of Lm90-△inlAB in mice＇s liver and spleen was much less than that of Lm 90 at different time points（tliver=2.454,P〈0.05;tspleen=2.443,P〈0.05）.Results reveal that the ability to resist stress of Lm 90 could have a significant change as genetic absence.Internalization InlA/InlB had a certain regulation effect on the ability of Listeria monocytogenes to infect host.Determination of biological characteristics of deletion strains provides a scientific basis for exploring Listeria monocytogenes survival under stress environment and pathogenic mechanism of virulence factors.