| Listeria monocytogenes(L.monocytogenes)is an important and worldwide food-borne pathogen.It has been listed as one of the four food-borne pathogens by the World Health Organization(WHO).Listeriosis is particularly associated with ‘at-risk’ consumers,predominantly affecting pregnant-women,older-adults(aged ≥ 60 years)and people receiving chemotherapy that causes severe illness(Septicemia,meningitis,abortion of pregnant women and neonatal death)with mortality rate as high as 30%.According to the distribution characteristics of L.monocytogenes isolates from food and humans in Shanghai,this study clarifies the differences of molecular subtyping,genetic and phenotypic characteristics of food and human isolates,which is of great significance for the prevention and control of Listeria monocytogenes.1.Serotype identification,molecular characterization and antimicrobial susceptibilityof 120 L.monocytogens isolatesA total of 120 L.monocytogenes isolates(107 from food and 13 from humans)were obtained from 2004 to 2013 in Shanghai.The food isolates included 30 from duck(25.00%),28 from chicken(23.00%)and 27 from beef(23.00%).Duck,chicken and beef were the high-risk foods for Listeriosis control.120 isolates belonged to three serotypes: 1/2a,3a(44 isolates),1/2b,3b,7(29 isolates),1/2c,3c(47 isolates).1/2a,3a(44 isolates),1/2b,3b,7(29 isolates)isolates have the potential risk of causing disease outbreaks and sporadic diseases.The proportion of 1/2c,3c serogroups of human isolates was only 7.69%.There were significant differences in the distribution of human and food sources.The difference was related to the nonsense mutation of inlA gene.A total of 74 PFGE patterns were generated among the 119 L.monocytogenes isolates(PFGE pattern of SHL12-70 was not gotten with Apa I).These PFGE patterns were grouped into two major branches,corresponding to lineages I and II,respectively.The isolates were grouped into 14 subgroups(S1-S14)with 80% pattern similarity.Subgroups S1 was the biggest subgroup that contained 1/2c.Three isolates from humans showed patterns similar to food isolates.This suggests that certain L.monocytogenes clones may remain and circulate locally within the Shanghai region of China.The same PFGE pattern was also shared by both human and food isolates collected from different years,indicating that those L.monocytogenes isolates causing listeriosis are associated with persistent contamination,and that consumption of raw or undercooked foods may be considered critical factors in the transmission of listeria foodborne infections.Drug resistance analysis of 14 antibiotics showed that 7 isolates were tetracycline resistant,13 ceftriaxone resistant.The drug resistance rate was 16.7%.Ceftriaxone is a first-line drug and is not recommended for the treatment of L.monocytogenes infection.Tetracycline-resistant isolates belong to 1/2a,3a serotype,carrying tet(M)resistance gene and transposon sequence of Tn916-Tn1545,which have potential risk of horizontal gene transfer.These isolates are distributed in many kinds of food.Combined with PFGE clustering analysis,cross-contamination can be identified among foods from different sources.2.Sequence characterization of virulence factor and inlA of L.monocytogenesThe distribution of 12 important virulence factors was detected using PCR assays.All isolates harbored hly,plcA,plcB,prfA,actA,inlA,inlB,and inlI.120 L.monocytogenes have potential pathogenicity.Sequence analysis of inlA gene,the key invasive factor,showed that there were 51 inlA nonsense mutants in 120 isolates,and 6 mutant types with regional characteristics.The nonsense mutants belong to two serotype groups: 1/2c,44 isolates of 3c,1/2a and 7 isolates of 3a.46.72% of the isolates from food sources carried the mutation,while only 7.69% of the isolates from human sources carried the mutation.There were differences in the distribution of the isolates from human sources and food sources.Phylogenetic development showed that the mutant inlA could be clustered according to the lineage type and serotype.3.Invasiveness Analysis of 21 L.monocytogenes and detecion of transcription level ofvirulence factorsThe 21 isolates were also examined for characteristics of biofilm formation and hemolysis.Invasion experiments were carried out using cell model Caco-2,Raw267.4 and animal model BALB/c.It was found that abilities of biofilm formation of 1/2c isolates was higher than that of the other two serotypes,which explained the widespread distribution of food-borne isolates of 1/2c.A significant difference(P <0.01)in the invasion between isolates with full-length and truncated inlA profiles was observed.The correlation between the existence of PMSC mutations in inlA and weak Caco-2 cell invading ability was found among the isolates examined(Pearson’s coefficient 0.927,p< 0.01)using SPSS analysis.Therefore,the mutation caused the decrease of invasiveness of isolates to break through the intestinal barrier.The strong invasive isolates(non-mutant isolates)and the weak invasive isolates(inlA non-sense mutant isolates)had the same invasive ability to Raw 267.4.There was no significant difference in the number of intracellular proliferations in 4 hours(p>0.05),which confirmed that L.monocytogenes had strong infective activity to phagocytes.RT-PCR was employed to analysis of the relative expression levels of virulence factors inlA,inlB,hly,BSH,prfA,hly of 21 sequencing isolates in BHI and chicken.The results showed that a relative higher-level expression of virulence factor shly and bsh were observed,inlA,inlB were expressed at medium level,and prfA expression was the poorest.There was no correlation between the relative expression of virulence factors and the insensitive mutant inlA gene carried by the isolates.Sequence analysis of five virulence factor promoters showed that the functional regions were highly conservative,which ensured the stable expression of virulence factors.Therefore,the isolate with inlA nonsense mutant still had potential pathogenicity.4.Genome sequencing analysis of L.monocytogenesHigh-throughput sequencing was performed on 21 L.monocytogenes isolates,including 13 human isolates and 8 food-borne isolates(7 non-sense inlA mutants).A total of 2247 core genes shared by 42 isolates(21 from the present study and 21 publicly available genomes)were selected,among which 153382 SNPs were identified and used to construct a maximum-likelihood phylogenetic tree.The 42 L.monocytogenes genomes were divided into two lineages based on phylogenetic data.Serotype1/2c isolates carrying PMSCs(Premature stop codons)in inlA were grouped together with nearly identical core genes sequences with SNPs ranging from 56 to 109.The genome sequencing data of the 21 isolates showed 14 isolates with full-length inlA and 7 isolates with six PMSC mutation types.Seventeen internalin genese quences were extracted from the 21 L.monocytogenes genomes.Whilst the other internalin genes were highly conserved compared with the polymorphic inlA gene,no PMSC mutations were found among these internalin gene sequences.New ST were identified in 7 isoaltes among 21 genome sequencing isolates,and others belonged to ST 3,7,8,9,29,87,381,391,753.Comparison on the sequences LIPI-/2/3 showed that there were differences among isolates with different serotypes.LIPI-2 of 1/2a isolate was composed of inlG,inlC2,inlD,inlE.LIPI-2 in 1/2b isolate was composed of inlC2,inlD,inlE.IPI-2 in 1/2b isolate was composed of inlG,inlH,inlE,inlG was carried only by Lineage II isolate.LIPI-3 was detected only in partial 1/2b isolate.The distribution of virulence islands was correlated with serotype. |
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