Introduction

Listeria monocytogenes (L. monocytogenes) is an important facultative intracellular bacterium that can cause severe invasive illness in humans with a high fatality rate [1]. L. monocytogenes is infectious to all humans. However, it has higher risk to cause severe diseases in the elderly, neonates, pregnant women, newborns, and immunocompromised individuals [23]. Clinical manifestations of listeriosis include meningitis, encephalitis, fetal loss, febrile gastroenteritis and septicemia [15, 18]. Contaminated foods are thought to be the primary source of listerial infections for both epidemic and sporadic cases, and the L. monocytogenes has been detected in variety of raw and readymade foods [8, 18, 19].

Since the strain characterizations of L. monocytogenes are necessary to epidemiological investigations and tracking individual strains in listeriosis outbreaks, a number of discriminatory subtyping methods have been developed for this organism. These methods include serotyping, pulsed-field gel electrophoresis (PFGE), multilocus enzyme electrophoresis (MLEE), ribotyping and multilocus sequence typing (MLST) [13]. Although the serotypes of L. monocytogenes described are as many as 13 types, more than 95 % of the strains isolated from contaminated foods and patients concentrate only on four serotypes, namely, 1/2a, 1/2b, 1/2c, and 4b [7, 20, 24]. Serotyping is widely used for long-term microbiological surveillance of human listeriosis and a more convenient multiplex PCR assay instead of traditional agglutination methods, was developed to distinguish the four major L. monocytogenes serotypes (1/2a, 1/2b, 1/2c, and 4b) corresponding to strains isolated from food and patients into various groups [4].

MLST based on nucleotide sequences of selected housekeeping genes is highly discriminatory to L. monocytogenes, with an advantage that it facilitates international comparison via MLST database contributed by all the researchers [17]. Previous investigations indicate that isolates of L. monocytogenes encompasses a great diversity of serotypes or genotypes with variegated virulent potential. Therefore, the evaluation of the pathogenic potential of isolates will contribute to the prevention from listeriosis and the improvement of food safety issues caused by L. monocytogenes. For assessing the virulence of L. monocytogenes isolates, the major techniques include serotyping or molecular typing, the detection of critical virulence factor, chicken embryos, the culture of cell lines in vitro, and mouse model in vivo [2, 5, 13, 16].

Although listeriosis is uncommon in China, L. monocytogenes has been isolated from dairy products, vegetables, poultry, pork, beef, and ready-to-eat food [10]. Recently, L. monocytogenes has been isolated from frozen crayfish tail meat and crayfish. However, there are few researches on virulence potential and polymorphism of L. monocytogenes strains isolated from freshwater live animals. In this study, the phylogenetic diversity and pathogenicity of L. monocytogenes crayfish-related isolates were assessed.

Materials and Methods

Bacterial Strains and Serotyping by Multiplex PCR

All isolates were cultured in Luria broth at 37 °C. L. monocytogenes were isolated from crayfish and the surveillance was generally performed through random sampling from open markets. The isolates were determined by PCR targeting hly and iap fragments specific for L. monocytogenes [25]. The multiplex PCR assay for serotyping was performed as described [4].

MLST Analysis

Genomic DNA of all the test isolates was extracted as previously described [12]. The MLST database used in this study is available at http://www.pasteur.fr/recherche/genopole/PF8/mlst/Lmono.html. Seven housekeeping genes acbZ, bglA, cat, dapE, dat, ldh, and lhkA were utilized as the target genes for MLST.

Based on the seven concatenated housekeeping gene sequences, neighbor-joining tree of L. monocytogenes isolates was constructed by using Molecular evolutionary genetics analysis software (MEGA 5.03) [22]. The program eBURST (v3.0) (http://eburst.mlst.net) was used to identify the different groups and clonal complex (CC). Discrimination index (DI), the ratio of nonsynonymous to synonymous substitutions (dN/dS), and the ratio of recombination to mutation (r/m) were calculated as described in previous study [9].

The Detection of Virulent Factors and Virulence to Mice

For each isolate, eight virulence genes prfA, mpl, actA, plcB, inlA, inlJ, virR, and mprF of L. monocytogenes were detected by PCR using the primer listed in Supplementary Table.

All the animal experimental protocols performed in the study were approved by the Laboratory Animal Monitoring Committee of Huazhong Agricultural University and performed accordingly. The animals were euthanized when moribund during the experiment or at the end of the experiments. 5-week-old Female ICR mice were used in this study as previously described [3].

Results and Discussion

Listeria monocytogenes is resistant to extreme pH, temperature, salt conditions [13], and even ionizing radiation [21]. These characteristics enable the bacterium to persist in raw material and food-processing, and make it an important human foodborne pathogen that may cause serious food safety problems. Thus, it is necessary to control the contamination before food-processing. Although a variety of surveillance on the genetic diversity and virulence of L. monocytogenes isolates from different sources, including chicken, pork, fish, and vegetables have been carried out, few studies on the isolates from crayfish have been reported so far.

In this study, 39 strains were isolated from live crayfish and further confirmed by PCR targeting hly and iap. More than 95 % of the L. monocytogenes strains isolated from contaminated foods and patients fall into serotypes 1/2a, 1/2b, 1/2c, and 4b [7, 20]. Multiplex PCR assay, which was developed to divide the four major L. monocytogenes serotypes (1/2a, 1/2b, 1/2c, and 4b) into distinct groups [4], was carried out to analyze the isolates from crayfish (Supplementary Fig). According to results of the amplifications of the four selected serotype-specific fragments, all the four serotype groups (1/2a, 1/2b, 1/2c, and 4b) were detected in this study, and the most frequent serotype group is 4b, 4d, 4e group with a frequency of 53.8 % (Table 1).

Table 1 Summaries of L. monocytogenes isolates in this study
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The 39 isolates from crayfish were divided into eight sequence types (STs) according to the MLST analysis, and the STs include ST1, ST4, ST87, ST9, ST122, ST155, ST35, and ST619. Six STs of the eight STs from crayfish had the same STs with the isolates from environment and clinic. ST1 caused the outbreaks of listeriosis in France in 1989 and in Sweden in 1995 [25]. ST8 and ST9 were the two most common STs isolated from Chinese food. ST619 has been only reported to be isolated from food in China. A certain number of the isolates shared the same STs with the clinical isolates, which suggested the strains isolated from crayfish are at a high risk of causing human infections.

The DI of each selected gene was calculated to compare the discriminatory power of each individual gene (Table 2). The DI values of the seven gene fragments ranged from 0.5107 for dapE to 0.6742 for dat. The most polymorphic gene is ldh with a DI value of 0.6629. The dat gene which had the most polymorphic sites than the other six housekeeping genes, exhibited a DI value of 0.6742. The data above indicated that the more polymorphic gene fragments or polymorphic sites, the better discriminatory power for subtyping. A few of STs were identified among the 39 isolates. The number of alleles within the seven housekeeping genes was small, ranging from 3 (lhkA) to 6 (ldh), while the number of polymorphic sites within the seven loci varied from 12 (bglA) to 58 (dat). The percentage of variable nucleotides per sites was greatest in dat (12.34 %). The ratio of nonsynonymous to synonymous substitutions (dN/dS) was low and this phenomenon usually happened in housekeeping genes while purified selection was in the dominate position. The ratio of recombination to mutation (r/m) was an important index to evaluate whether recombination or mutation was the key point to determine the heterogeneous relationship. In the present study, mutation played greater important role in determining the genetic diversity than recombination, since the ratio of r/m of all loci was much lower than 1.

Table 2 Allelic polymorphisms of seven selected genes fragments
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The eBURST v3.0 program divided the eight STs into one CC and five singletons. The CC consisted of ST9, ST35, and ST122, while ST9 was predicted to be the ancestry (Fig. 1a). Of the 39 strains, only eight STs were identified, and only one CC was formed, which revealed the conservative evolutionary relationship between the strains isolated from crayfish. In order to demonstrate the clustering and evolutionary model constructed through eBURST, a neighbor-joining tree representing the concatenated sequences of the seven HK genes fragments in 39 isolates is presented in Fig. 1b. As shown in the figure, the strains with the same ST were grouped into same branches. And the strains belonging to the same CC were also grouped together. However, what neighbor-joining tree revealed was slightly different from what eBURST presented, for instance, ST1 and ST35 were clustered into one clade whereas they were not in the same group or CC by eBURST. Another neighboring-joining tree was constructed to make a brief comparison between the strains isolated from this study and clinical strains isolated from China in the database. Some isolates from crayfish shared the same ST with isolates from patients, while ST381 and ST303 were clinical isolates and these two STs and the CC were grouped into the same branch. This suggested it was possible for the strains belonged to the CC to infect humans.

Fig. 1
figure 1

Genetic relationships of the isolates based on MLST. a The popular structure of the eight STs isolated from crayfish. The size of the circle is proportional to the number of the isolates. b Neighboring-joining tree of 39 L. monocytogenes isolates, the tree was constructed using the concatenate nucleotide sequences of seven housekeeping genes. c Neighboring-joining tree of 39 crayfish isolates, four clinical isolates, and six fish isolates

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Targeting the virulence-associated genes constitutes a convenient way to determine virulence of L. monocytogenes in practical surveillance. InlJ (lmo2821) is considered as a sensitive target of distinguishing virulent from avirulent isolates. lmo2821 is present in L. monocytogenes isolates causing human listerial outbreaks but absent in avirulent or non-pathogenic isolates. Thus, the detection of the genes related to L. monocytogenes virulence, such as prfA, mpl actA, plcB, inlA, inlJ, virR, and mprF is important for understanding the virulence of L. monocytogenes [6, 11, 14, 23]. The PCR screening results showed that all the isolates from crayfish contained eight important virulent factors (Fig. 2a). In contrast, most of the strains isolated from food products in previous study only contain part of the virulence-associated genes [25].

Fig. 2
figure 2

Virulence of L. monocytogenes isolates. a PCR determination of eight virulent factors in isolates. b Survival curve of ST1, ST4, and ST9 isolates in murine infection model

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Of the methods mentioned above, only murine model in virulence assay provides an in vivo assessment of isolates and is often considered as a standard for testing virulence of L. monocytogenes. To evaluate the virulence of the isolates, each strain from ST1, ST4, and ST9 was randomly chosen for mice experiment in vivo. All the mice in the infected groups, including the mice infected with the virulent strain EGDe (positive control), were found to suffer from illness in 2 h and all died within 12 h at the dose of 109 CFU by intraperitoneal injection (data not shown). At the dose of 108 CFU, all the infected mice die within 40 h, and the survival curve of each strain was recorded (Fig. 2b). The survival curve of each isolates showed no significant difference with that of the virulent strain EGDe, which indicated the isolates have the potential to cause human disease. Although human listeriosis was uncommon in China and there have been few reports on the outbreaks of human listeriosis so far, surveillance is still needed to control the potential risk for the sake of food safety.