Molecular Subtyping Of Listeria Monocytogenes Isolates From Different Sources

Listeria monocytogenes(L.monocytogenes) is an important pathogen that is responsible for listeriosis in humans and animals. Listeriosis is a very severe disease which may cause meningitis, sepsis and abortion, with a mortality rate in humans of20%to30%. It is one of the most deadly bacterial infections currently known. L.monocytogenes infection of animals may be mainly caused by feeding of contaminated silage, while human listeriosis is caused by food contaminated by L.monocytogenes. Human listeriosis had outbroken in many European and American countries, some of which were caused by animal products. The listeriosis outbreaks related to animals and humans were also occurred in China. Many countries have paid attention to L.monocytogenes because of its serious threat to public health.The prevalence of L. monocytogenes was invesitigated in eastern China. Multilocus aequence typing (MLST) was used to study the evolution and phylogeny of isolates from human, animal and food. The population structure, virulence evolution and invasiveness were studied with lineage classification and Multi-Virulence-Locus Sequence Typing (MVLST) based on virulence and virulence-associated genes, as well as infection assay to mice.1. Isolation and identification of L.monocytogenes in foodThe prevalence of L.monocytogenes in six kinds of food (pork, chicken, beef, ready-to-eat food and seafood) was detected. Food samples were incubated in Listeria Enrichment Broth, followed by streaked on CHROMagarTM Listeria. Identification of Listeria spp. and their species was carried out using multiplex PCR. The serotypes of L.monocytogenes isolates were identified with antisera and multiplex PCR.93L.monocytogenes,150L.innocua and4L.welshimeri were isolated from538samples. The isolation rate of L.monocytogenes was17.29%. There were46 serotype1/2a strains,16serotype1/2b strains,30serotype1/2c strains and1serotype4b strains. The results showed that the prevalence of L.monocytogenes in11cities was high. Most isolates were serotype1/2a,1/2b and1/2c. Cold fresh and frozen food had a significantly higher isolation rate of L.monocytogenes compared to room temperature food (P<0.05). Raw meat such as pork, beef and chicken were contaminated by L. monocytogenes more seriously than other kinds of food. L.monocytogenes causes threat to food safety. It should pay more attention to food processing, transport and retailing.2. Multilocus Sequence Typing analysis of L.monocytogenes isolates from different sourcesMLST makes use of automated DNA sequencing to characterize the alleles present at different housekeeping genes. These genes are slowly diversified, which is useful for studying the population structure and evolution of L.monocytogenes. Minimum spanning tree based on sequence type (ST) was structured to study the population structure of isolates from different sources and serotypes. The64isolates were divided into25STs, including three new STs (ST618、ST619and ST626). Three worldwide spread clonal complexs (CCs) were identified, including CC1, CC3and CC9. The most common STs were ST3, ST5, ST9and ST87. Many STs contained iaolates from human and animal clinical listeriosis. CC9contained isolates from humans, animals and food. In general, each ST had a unique serotype, while ST1, ST3and ST9contained many serotypes. According to the minimum spanning tree, serotype1/2a and1/2c were genetic closely, while serotype4b,1/2b,4d and4e were genetic closely. On the evolution branch, serotype1/2b and4b showed a unique "intervel-type" evolution, while serotype1/2a and1/2c showed a "progressive type" evolution. Considering, the population structure of isolates from food and STs or CCs that are over-represent among isolates from humans, animals and food. In addition, the evolution of different serotypes was further studied by Minimum Spanning Tree based on MLST analysis.3. Molecular subtyping based on sequencing of virulence and virulence-associated genesThe lineage classification of isolates from human, animal and food was studied by cluster analysis of3’end partial sequence of virulence gene actA. Isolates were classified into four lineages (lineage Ⅰ, Ⅱ, Ⅲ, and Ⅳ). Most isolates were divided into lineage Ⅰ and Ⅱ. Lineage Ⅰ included serotype4b,1/2b,4d and4e, while serotype1/2a and1/2c belonged to lineage Ⅱ. Lineage Ⅰ, Ⅲ, and IV contained serotype4b. These four evolutionary lineages (Ⅰ, Ⅱ, Ⅲ, and Ⅳ) have different ecological, genetic and phenotypic characteristics, which is closely related to the ability to be transmitted through food and to cause listeriosis.Because of limited sequence variation in housekeeping genes, MLST sometimes lacks the discriminatory power required to address issues of local epidemiology and some serotypes. The evolution and pathogenicity of isolates were evaluated by MVLST (Multi-Virulence-Locus Sequence Typing) and invasion assay on mice. Serotype1/2b and4b were divided into two clusters, Cluster A and Cluster B respectively. Furthermore, serotype4b isolates were divided into two subclusters. One of the subclusters was serotype4b isolates that the majority were isolated from food except one from human clinical case. Another subcluster contained serotype4d/4e and1/2b, as well as serotype4b. Some human and animal listeriosis outbreaks isolates belonged to this subcluster. MVLST did not separate serotype1/2a and1/2c isolates into two distinct clusters, but all serotype1/2c isolates were in the same cluster, which indicated that serotype1/2a and1/2c were highly clonal. The MVLST analysis showed the differences of virulence gene evolution between different serotypes and in the same serotype. Invasion ability of isolates was determined on BALB/c mice via intravenous injection route, the result showed that different serotypes had different invasiveness and the pathogenicity was different. The invasiveness of serotype1/2a was significantly higher than serotype1/2b and4b. The results of MLST, lineage classification and MVLST indicated that there was some degree of evolution consistency between housekeeping genes, virulence and virulence associated genes, as well as serotype antigen genes.In summary, the prevalence of L.monocytogenes in food was demonstrated in Eastern China. MLST showed the phylogeny of isolates and the epidemic STs or CCs in food. MVLST indicated the differences of virulence gene evolution between different serotypes. This study would be useful for provided new insight to L.monocytogenes and prevention and control of listeriosis.