Listeria characteristics

Background

Ready-to-eat [RTE] vegetables have become increasingly popular along with the trend of moving towards a healthy lifestyle. However, RTE vegetables are at a higher risk of containing pathogens, maybe owing to lack of rigorous sanitization procedures. To understand the prevalence and potential risk of Listeria monocytogenes in RTE vegetables, we investigated the contamination level and characteristics of L. monocytogenes isolated from fresh vegetables.

Results

Twenty-three [5.49%] of the 419 vegetables samples were positive for L. monocytogenes. Phylogenetic group I.1 [1/2a-3a] and II.2 [1/2b-3b-7] strains were predominant in 30 isolates, which accounted for 33.3 and 50.0%, respectively. Multilocus sequence typing of the 30 isolates grouped them into nine sequence types [STs]. The most common STs were ST87 [36.7%] and ST8 [26.7%]. Virulence analysis showed that all 30 isolates harbored eight classical virulence genes, 10.0% isolates harbored the llsX gene [ST3 and ST1 strains], and 36.7% carried the ptsA gene and belonged to ST87. Approximately 83.3% isolates carried full-length inlA, whereas five isolates had premature stop codons in inlA, three of which belonged to ST9 and two to ST8. Antibiotic susceptibility showed the isolates were varyingly resistant to 13 antibiotics, 26.7% of the isolates were multi-drug resistant.

Conclusions

The fresh vegetables contain some potential hypervirulent L. monocytogenes [ST1 and ST87] in the Chinese markets. In addition, the high rate of L. monocytogenes isolates was multi-drug resistant. Fresh raw vegetables may be a possible transmission route for L. monocytogenes infection in consumers. Therefore, sanitization of raw fresh vegetables should be strengthened to ensure their microbiological safety when used as RTE vegetables.

In recent years, there is an increasing demand for ready-to-eat [RTE] vegetables because people have realized the importance of a healthy diet and healthy lifestyle. However, the increasing consumption of fresh produce, including fruits and vegetables was recognized as a source of microbiological foodborne outbreaks in many parts of the world [1, 2].

Listeria monocytogenes is a common Gram-positive facultative anaerobic bacillus that is recognized as one of four foodborne pathogens by the World Health Organization. It can invasively infect humans and animals, causing severe listeriosis leading to meningoencephalitis, sepsis and fetal infection or miscarriage in pregnant women, with a mortality rate of 2030% [3]. High-risk groups include pregnant women, infants, immunodeficient patients and the elderly [3]. Fresh vegetables and herbs, such as coriander, lettuce, tomato, cucumber, cabbage, carrot, and shallot, are generally used as raw material in RTE vegetables after minimal processing to preserve nutrients, which is an important source of RTE vegetable pollution. In recent years, reports show an increase in the prevalence of L. monocytogenes in fresh and RTE vegetables. A study revealed that L. monocytogenes was detected in 6.67% fruit and vegetable products from 2011 to 2015 in Jilin Province, China [Yang et al., 2017]. Both in Europe and USA, a link between foodborne disease outbreaks and pathogen [such as L. monocytogenes] contaminated green leafy vegetables, such as lettuce and spinach, and their RTE salads has been reported [4]. In recent years, the occurrence of L. monocytogenes in vegetables has been reported in several countries, with the detection rate ranging from 0.6 to 9.1% [5,6,7,8]. China is the largest vegetable producer and consumer in the world, with an output volume of vegetables and processed products of 10.1 million tons in 2015 [9], However, there is no uniform method for risk assessment and management of L. monocytogenes in RTE vegetables [10],which are mostly consumed raw or after minimal processing and pathogen contamination poses a health risk.

It is critical to evaluate the virulence of L. monocytogenes in fresh vegetables. This study aimed to evaluate the qualitative and quantitative contamination level by L. monocytogenes in fresh vegetables from Chinese markets. We determined the phylogenetic group, virulence profiles, and antimicrobial resistance and performed multilocus sequence typing [MLST] to comprehensively study the virulence of L. monocytogenes isolated from fresh vegetables across China.

Qualitative and quantitative analysis of L. monocytogenes in fresh vegetable samples

A total of 23 [5.49%] positive samples were detected by quantitative and qualitative methods in the 419 collected vegetable samples. According to the different types of vegetables collected, the positive rate of lettuce was highest [7.78%], followed by coriander [4.49%], tomato [4.90%] and cucumber [4.88%] [Table 1]. Among the 23 positive samples, quantitative detection by MPN method showed that the contamination level of two samples corresponding to lettuce and tomato were up to 110 MPN/g, one tomato sample was 24 MPN/g, and the remaining 20 samples were between 0.310 MPN/g. We also isolated 30 strains of L. monocytogenes from the 23 positive samples [Additionalfile1: Table S2].

Phylogenetic group analysis

Multiple PCR was used to analyze the phylogenetic group of 30 isolates obtained from 23 L. monocytogenes-positive samples [Additional file 1: Figure S1]. Isolates belonging to different phylogenetic group from the same sample were included in this study. Isolates were divided into four phylogenetic groups, among which phylogenetic group I.1 [1/2a-3a] accounted for 33.3% [10/30], phylogenetic group I.2 [1/2c-3c] accounted for 13.3% [4/30], phylogenetic group II.1 [4b-4d-4e] accounted for 3.3% [1/30], and phylogenetic group II.2 [1/2b-3b-7] was predominant at 50.0% [15/30] [Fig.1]. L. monocytogenes of two different phylogenetic groups were isolated from three lettuce samples, one cucumber sample and one coriander sample, suggesting that there were different serotypes of L. monocytogenes contaminating the same vegetable sample.

Multilocus sequence typing analysis

According to the sequencing results of the seven housekeeping genes using MLST, the sequence types [STs] and clonal complexes [CCs] of the isolates were obtained. The 30 isolates were classified into nine STs belonging to nine CCs [Fig. 1]. ST87/CC87 and ST8/CC8 were predominant and accounted for 36.7% [11/30] and 26.7% [8/30], respectively. CC9 accounted for 13.3% [4/30], CC3 for 6.7% [2/30], and CC1, CC5, CC7, CC59 and CC155 were 3.3% [1/30] each, respectively.

Virulence profiles analysis

In this study, a total of 10 virulence genes [prfA, mpl, plcA, inlB, plcA, hly, iap, actA, llsX and ptsA] of 30 isolates were detected by PCR. The results showed that except for the llsX and ptsA, all isolates carried the other eight virulence genes. Only three isolates [413-2LM, 1965-1LM and 2013-1LM] carried the llsX gene, which belonged to ST1 and ST3. A total of 11 [36.7%] isolates harbored the ptsA gene and all belonged to ST87 [Additional file 1: Figure S2]. Analysis of inlA DNA sequencing showed that five isolates harbored PMSCs in the inlA gene. Four isolates [2114-1LM, 2114-3LM, 2363-1LM and 2363-3LM] had a deletion of an adenine at position 1637 of inlA [11], and one isolate [3015-1LM] had a deletion of an adenine at position 12 [12], These frame shift mutations lead to the creation of a nonsense codon, TAA, at position 1729 and 25, respectively. Moreover, three isolates [2114-1LM, 2363-1LM, and 2363-3LM] belonged to ST9, and the other two [3015-1LM and 2114-3LM] belonged to ST8.

Antimicrobial resistance analysis

According to CLSI guidelines and MIC method, the antimicrobial resistance analysis of 16 antibiotic agents against the L. monocytogenes isolates is shown in Table2 and Fig. 1. Among the 30 L. monocytogenes isolates, all strains were susceptible to three antibiotics, penicillin, ampicillin, and sulfamethoxazole/trimethoprim. In contrast, resistance to clindamycin was the most common resistance and was identified in 63.3% isolates. On the other hand, only four [13.3%] isolates [133-1LM, 413-2LM, 413-4LM, and 1741-1LM] were susceptible to all the 16 antibiotics. Other isolates showed varying degrees of resistance and/or intermediate resistance to individual antibiotics. It is worth mentioning that 26.7% isolates were defined as multi-drug resistant, of which 1331-1LM and 1331-2LM were isolated from the same tomato sample and showed resistant/intermediate resistant to 12 and 9 antibiotics, respectively.

There is no doubt that L. monocytogenes is a critical foodborne pathogen worldwide. At present, there are very little data on the identification of L. monocytogenes contamination in fresh vegetables in China, especially for quantitative analysis, which limits risk assessment and the development of relevant safety standards for fresh vegetables used in RTE vegetables. In this study, 419 commercially available fresh vegetables in 43 representative cities/regions in China were investigated, of which 23 [5.49%] were positive for L. monocytogenes [Table 1]. The highest contamination rate [7.78%] was found in lettuce sample and the MPN value of one positive sample exceeded 100 MPN/g. This could be due to the relatively large leaf surface of lettuce that is close to the soil surface and therefore easily contaminated by soil and environmental water. Wang et al. analyzed 153 RTE vegetable samples from six districts in Zigong City, Sichuan Province of China, and found a L. monocytogenes positive rate of 6.5% [13], which was consistent with our results. Kuanet al. reported that L. monocytogenes was more frequently observed in organic [9.1%] than in conventional [2.7%] vegetables in Malaysia [6]. These studies have reported different degrees of L. monocytogenes contamination in RTE vegetables. To date, National Health Commission of P. R. China has formulated a zero-tolerance policy for L. monocytogenes in cooked meat and ready-to-eat fresh meat. European Union has formulated a