北京市朝阳区单核细胞增生李斯特菌关键毒力基因缺失与致病性关联性研究

Study of association between deletion of key virulence genes and pathogenicity of Listeria monocytogenes in Chaoyang District of Beijing

目的了解北京市朝阳区2016—2018年81株单核细胞增生李斯特菌(单增李斯特菌)关键毒力基因缺失与菌株致病性和血清型的关联性。方法聚合酶链反应(PCR)结合血清凝集法进行血清学分型;PCR法检测11种毒力基因;微量肉汤稀释法进行药敏实验;结合流行病学调查数据,研究单增李斯特菌关键毒力基因缺失与致病性、血清型的关联。结果81株单增李斯特菌分为4种血清型:1/2a型占50.62%(41/81)、1/2b型占29.63%(24/81)、1/2c型占11.11%(9/81)、4b型占8.64%(7/81);食源性菌株与病例分离株优势血清型均为1/2a。毒力基因inlA、inlC、inlJ、plcB、prfA、mpl携带率为100%;iap、hly、actA、inlB、plcA携带率分别为98.77%、98.77%、97.53%、80.25%、38.27%;携带7种、8种、9种、10种、11种毒力基因的菌株比例分别为1.23%(1/81)、2.47%(2/81)、7.41%(6/81)、59.26%(48/81)、29.63%(24/81)。hly在1/2c型低于其他血清型,plcA仅在1/2b和4b型中100%携带。70.37%(57/81)的单增李斯特菌株存在毒力基因缺失,1/2a型为主(41/57,71.93%);1/2b型(6/24,25.00%)与4b型(1/7,14.29%)仅缺失inlB。病例分离株中的16株(16/28,57.14%)毒力基因缺失株,性别、年龄、临床菌株来源以及是否妊娠相关的分布差异均无统计学意义(P>0.05)。81株单增李斯特菌对青霉素、红霉素、氨苄西林的耐药率均为4.94%,对其他抗生素最低抑菌浓度(MIC)值较小,但有57株单增李斯特菌对氯霉素MIC值达16μg/mL;毒力基因缺失株存在3株耐药菌株,一株为inlB缺失株,对青霉素、红霉素、氨苄西林均耐药;另一株为inlB缺失株仅对青霉素耐药;第三株plcA缺失株对红霉素耐药。结论北京市朝阳区单增李斯特菌食源性菌株与病例分离株血清型以1/2a型为主,存在毒力基因缺失现象,但部分毒力基因缺失对菌株致病性影响有限,食品和临床分离单增李斯特菌的耐药率偏低,但应加强单增李斯特菌毒力基因和耐药性的监测与防控。

Objective To investigate the association between deletion of key virulence genes and pathogenicity and serotype of 81 strains of Listeria monocytogenes(L.monocytogens)isolates in Chaoyang district of Beijing from 2016-2018.Methods Serotyping was performed on 81 strains of L.monocytogenes by polymerase chain reaction(PCR)and serum agglutination assay,and 11 virulence genes(inlA,inlC,inlJ,plcB,prfA,mpl,iap,hly,actA,inlB,plcA)were detected by PCR.The susceptibility of these strains to 9 antibiotics were tested by broth dilution.Based on the epidemiological survey data,the relationship between the deletion of key virulence genes,pathogenicity and serotype of L.monocytogenes was studied.Results The 81 strains of L.monocytogenes were classified into 4 serotypes:1/2 a serotype 50.62%(41/81),1/2 b serotype 29.63%(24/81),1/2 c serotype 11.11%(9/81)and 4 b serotype 8.64%(7/81).The predominant serotype of food isolates and patient isolates were 1/2 a.The positive rates of virulence genes inlA,inlC,inlJ,plcB,prfA and mpl were 100%.The positive rates of iap,hly,actA,inlB and plcA were 98.77%,98.77%,97.53%,80.25%and 38.27%respectively.The proportions of the 81 strains of L.monocytogenes carrying 7,8,9,10 and 11 virulence genes were 1.23%(1/81),2.47%(2/81),7.41%(6/81),59.26%(48/81)and 29.63%(24/81).The positive rate of hly in genotype 1/2 c was lower than that in other serotypes,and plcA was only 100%carried in genotype 1/2 b and 4 b.70.37%(57/81)isolates lacked 11 virulence genes and 1/2 a(41/57,71.93%)was the main serotype.Only inlB was not detected in 1/2 b(6/24,25.00%)and 4 b(1/7,14.29%)serotype.There was no significant difference in the distribution of gender,age,pregnancy related or not,and the source of strains in 16 virulence gene deletion strains isolated from patients(P>0.05).The resistant rates of 81 strains of L.monocytogenes to penicillin,erythromycin and ampicillin were 4.94%.MIC to chloramphenicol of 57 strains was as high as 16μg/mL and MIC to the other antibiotics was low.There were three drug-resistant strains in the virulence gene deletion strains,and one inlB deletion strain was resistant to penicillin,erythromycin and ampicillin.Another inlB deletion strain was resistant to penicillin.The plcA deletion strain was resistant to erythromycin.Conclusion The dominant serotypes of L.monocytogenes isolated from food and patients in Chaoyang district of Beijing was 1/2 a.There were some virulence gene deletion strains.Deletion of some virulence genes had limited effect on pathogenicity of L.monocytogenes strains.The drug resistance rate of L.monocytogenes isolated from food and clinic was low,but surveillance and control of virulence genes and drug resistance of L.monocytogenes should be strengthened.