Listeria monocytogenes is a major foodborne pathogen causing human listeriosis, a severe foodborne disease with high hospitalization rates (97.0%) and mortality rates (15.6%) (European Food Safety Authority [EFSA], and European Centre for Disease Prevention and Control [ECDC], 2019). Although L. monocytogenes accounted for 1% of foodborne disease outbreaks, it caused the highest percentage of foodborne disease-related deaths in the United States from 2009 to 2015 (Dewey-Mattia et al., 2018). The infection of L. monocytogenes involves the elderly, immunocompromised people, newborns, and pregnant women, which leads to sepsis, meningitis, encephalitis, abortion, and stillbirth (Lomonaco et al., 2009; Camargo et al., 2019). Its ability to survive and grow under a wide range of environmental conditions, including refrigeration temperatures, high salt concentrations, and low pH, makes it a potential hazard in foods (Ryser and Marth, 2007; Wu et al., 2016). L. monocytogenes causes listeriosis, with most cases caused by consuming contaminated food (Pérez-Rodríguez et al., 2017; El-Hajjaji et al., 2021). Meat, milk, and vegetables represent important sources of contamination, and L. monocytogenes can survive and multiply in raw products or ready-to-eat food made with such raw items, even if refrigerated (Filiousis et al., 2009).

Edible mushrooms, such as Lentinula edodes, Auricularia auricula-judae, Pleurotus ostreatus, Flammulina velutipes, Pleurotus eryngii, Agaricus bisporus, and Auricularia polytricha, are popular worldwide according to their taste and nutritional value. In 2020, the total output was 40.6 million tons in China, with an economic output of RMB ¥346.6 billion, accounting for ∼75% of global mushroom production. However, contaminations detected in food processing plants have led to rigid recalls of products and serious economic losses. In recent years, several recalls have occurred after detectable levels of L. monocytogenes were found on whole or sliced fresh mushrooms (Viswanath et al., 2013). More recently, L. monocytogenes was found contaminated in enoki mushrooms, which are responsible for 36 reported cases of listeriosis, including 31 hospitalizations and four deaths in the United States (Byun et al., 2022). Meanwhile, the occurrence of L. monocytogenes in edible mushroom products has been reported in several countries (Cordano and Jacquet, 2009; Venturini et al., 2011; Viswanath et al., 2013; Chen et al., 2018). However, it is necessary to understand how L. monocytogenes is entering mushroom production facilities through incoming raw materials and/or surviving and growing in farm and packing house environments.

Different molecular typing methods have been employed to assess the source of contamination and the routes of transmission of L. monocytogenes. The multilocus sequence typing (MLST) method is one of the most robust tools for investigating the global epidemiology of microbial populations (Sullivan et al., 2005). In addition, differentiation between virulent and hypo-virulent strains is significant for evaluating the potential implications of the presence of this microorganism for food safety and public health (Jensen et al., 2008). Among the 14 serotypes of L. monocytogenes identified, the results of multiplex polymerase chain reaction (PCR) indicated that these major serogroups could be separated into distinct groups: I.1 (1/2a-3a), I.2 (1/2c-3c), II.1 (4b-4d-4e), II.2 (1/2b-3b-7), and III (4a-4c) (Doumith et al., 2004). Serotypes 4b, 1/2a, and 1/2b are mostly associated with listeriosis in humans (Orsi et al., 2011; Poimenidou et al., 2018; Pérez-Baltar et al., 2021). Moreover, many virulence traits that participate in host invasion and cellular proliferation are mainly distributed in Listeria pathogenicity islands (LIPI), which have been identified depending on the pathogenicity of the strain and severity of the infection (Radoshevich and Cossart, 2018; Al et al., 2022). The prfA-regulated virulence gene cluster (pVGC), which consists of a monocistronic hly in the center of the locus, a lecithinase operon comprising mpl, actA, and plcB genes, and the plcA-prfA operon located upstream from hly and transcribed in the reverse direction are often referred to as Listeria pathogenicity island 1 (LIPI-1) (Vázquez-Boland et al., 2001a; Ward et al., 2004; Poimenidou et al., 2018). In addition, the internalins (InlA, InlB, InlC, and InlJ) play key roles in adhesion and invasion, which are essential for the pathogen to invade intestinal cells (Bonazzi et al., 2009). Furthermore, the determination of the genetic profile and antimicrobial susceptibility profile of L. monocytogenes isolates present in food sold in open markets may also help to trace future listeriosis outbreaks.

In a previous study, it was demonstrated that various retail edible mushroom samples such as F. velutipes, P. eryngii, Volvariella volvacea, Hypsizygus marmoreus, Pleurotus geesteranus, P. ostreatus, and L. edodes could both contaminate L. monocytogenes (Chen et al., 2018). Few other studies explored the contamination sources in F. velutipes, H. marmoreus, or other edible mushroom production facilities (Viswanath et al., 2013; Chen et al., 2014; Murugesan et al., 2015; Sun et al., 2021), but in P. eryngii, the situation is unclear. Considering that the processing of production for P. eryngii was similar to that of F. velutipes or H. marmoreus, this study was designed to investigate the prevalence of L. monocytogenes in four P. eryngii production plants and processing environments to explore the contamination sources and potential risk of the L. monocytogenes isolates in P. eryngii based on their genotypes determined by molecular serotyping and MLST, their antibiotic susceptibility, and their virulence gene profiles.

In recent years, the contamination of L. monocytogenes has been found in edible mushrooms. Although few listeriosis outbreaks attributed to fresh mushrooms have been reported, recalls of mushroom products are common, caused by contamination with L. monocytogenes (Sun et al., 2021). P. eryngii, also known as the king oyster mushroom or cardoncello, is becoming increasingly popular with consumers in Europe, Asia, and North America. In recent times, it has become one of the most important commercial producers and exporters of edible mushrooms in China. However, the contamination source of L. monocytogenes in P. eryngii remains unclear. In this study, we investigated the prevalence of L. monocytogenes in four P. eryngii production plants from four cities and found that 14.3% of samples were positive for L. monocytogenes, including 13.3% of mushroom samples and 14.7% of environmental samples. The contamination is likely to originate from the mycelium stimulation machine, which coincides with previous research in the F. velutipes and H. marmoreus production facilities (Chen et al., 2014; Sun et al., 2021). In addition, the prevalence rate of L. monocytogenes in the P. eryngii production facility (14.3%) was higher than the A. bisporus production facility (1.6%) (Viswanath et al., 2013) but lower than the H. marmoreus production facility (27.0%) and the F. velutipes production facility (18.6%) (Chen et al., 2014). Compared with the mushroom samples (including the growing stage, harvesting stage, and products), our results were similar to those of H. marmoreus but lower than those of retail F. velutipes products (55.5%) (Chen et al., 2018). To our knowledge, the fruiting temperature of F. velutipes (6–12°C) is lower than that of P. eryngii (10–18°C) and H. marmoreus (12–16°C) in modern industrial cultivation of edible mushrooms. When L. monocytogenes was exposed to or persisted in the associated environment, its ability to proliferate at low temperatures may also play a key role in the persistence of this bacterium in harvesting and refrigerating stage product. Thus, the temperature accompanied by cross-contamination was probably the major reason for L. monocytogenes contamination in edible mushrooms.

As we know, different molecular typing methods have been employed to assess the source of contamination and the routes of transmission of L. monocytogenes. In this study, MLST was used and proved to be particularly suitable for the epidemiology investigations of food processing for L. monocytogenes. Overall, the source of L. monocytogenes contamination in the P. eryngii production plant was different and occurred inside the plant. Except for one isolate (A2601LM) belonging to ST9 from plant A, the isolates of L. monocytogenes focus on ST121 and ST3, which are found in plants A and B and originate from mycelium stimulation machines. The contamination was focused on P. eryngii production plants A and B, which are likely to originate from the mycelium stimulation stage. Meanwhile, in plants C and D, isolates from various STs (ST9, ST224, and ST87) were isolated from surfaces, floors, or drains, respectively. Thus, the L. monocytogenes contamination in the P. eryngii production plant could be caused by a wide variety of environments, but the contamination of the mycelium stimulation machine was the most important factor in this study. This is consistent with previous research in the F. velutipes and H. marmoreus production facility, which showed that the mycelium-scraping machine was the main transmission source in the production process (Chen et al., 2014; Sun et al., 2021).

Furthermore, the contamination of L. monocytogenes may also persist in P. eryngii production plants. Persistence has been defined as “repeated isolation on different dates of L. monocytogenes strains that are identified as identical subtypes (as determined by phenotypic or genotypic methods)” (Ferreira et al., 2014; Maury et al., 2016). According to previous studies, the L. monocytogenes ST121 strains, also found in this study, have previously been isolated from food and food processing facilities over several years in processing plants (Hein et al., 2011; Holch et al., 2013; Stoller et al., 2019). In Hein et al. (2011) found that the ST121 strains were isolated in Austria and Belgium from different ecological niches, including food and human cases, over several years. Similarly, Holch et al. (2013) reported that the ST121 strains isolated from four of eight different Danish fish processing industries were the persistent and dominant type in three plants over a period of 6 years, and Stoller et al. (2019) found that L. monocytogenes ST121 strains persisted in a meat processing facility over a 4-year period. According to the research of Harter et al. (2017), L. monocytogenes ST121 strains have the stress survival islet 2 (SSI-2) that is beneficial for survival under alkaline and oxidative stress conditions and is commonly encountered in food processing environments (Harter et al., 2017). All L. monocytogenes ST121 strains isolated from our study occurred PMSC mutations in inlA, which indicates that the ability of these persistent strains to attach to human host cells may decrease and also virulence attenuation (Van Stelten and Nightingale, 2008). Except for ST121, we also found ST3 isolates persisting in P. eryngii production plant B. These isolates belonged to serogroup II.1 (4b, 4d, and 4e), which was mostly related to food or humans with listeriosis (Sosnowski et al., 2019). According to the population structure analysis of L. monocytogenes isolates in Australia from 1931 to 2015, ST1 and ST3 were the most abundant strains (Jennison et al., 2017). In Poland, ST3 was the second most common type in L. monocytogenes isolates from various types of food of animal origin during 2013–2016 (Sosnowski et al., 2019). CC3 is the fourth most common clonal complex in Europe, is predominant in Australia, and was also detected in South and North America, Japan, and Oceania (Bespalova et al., 2021). Thus, the persistence of ST3 L. monocytogenes in the P. eryngii production plant deserves published attention. However, the reasons why ST3 L. monocytogenes persist in P. eryngii production plants remain to be further explored.

Listeria monocytogenes is classified into at least four evolutionary lineages (I, II, III, and IV) with different serotypes. Isolates of serotypes 1/2b, 3b, 3c, and 4b were assigned to lineage I, whereas isolates of serotypes 1/2a, 3a, and 1/2c were assigned to lineage II, and isolates of serotypes 4a and 4c were assigned to lineage III (Orsi et al., 2011). In this study, the L. monocytogenes isolates were serogroup II.1 (4b, 4d, and 4e), serogroup I.1 (1/2a and 3a), and serogroup I.2 (1/2c and 3c), which both belonged to lineage I and lineage II. As previously studied, most L. monocytogenes isolates belong to lineages I and II, which harbor the serotypes more commonly associated with human clinical cases, including serotype 1/2a (lineage II) and serotypes 1/2b and 4b (lineage I) (Orsi et al., 2011). Lineage I strains are mostly associated with the majority of human listeriosis outbreaks worldwide (Aureli et al., 2000; Jeffers et al., 2001; Gray et al., 2004), whereas lineage II strains are common in foods, seem to be widespread in natural and farm environments, and are also commonly isolated from animal listeriosis cases and sporadic human clinical cases (Lukinmaa et al., 2003; Parihar et al., 2008). Although L. monocytogenes has 14 serotypes that differ in virulence potential, serotypes 3a, 3b, 3c, 4a, 4c, 4e, and seven are very infrequent in food (Kathariou, 2002; Doumith et al., 2004; Yin et al., 2019). Thus, the dominant serotypes of L. monocytogenes isolated from the P. eryngii production facility should be 4b (15/29, 51.4%) and 1/2a (10/29, 34.5%), whereas 1/2c (4/29, 13.8%) is limited. However, the serotypes of L. monocytogenes isolates found in P. eryngii production plants have a close connection with human listeriosis outbreaks and should receive much attention.

In this study, the virulence genes, such as hly, inlB, inlA, inlC, inlJ, actA, iap, plcA, mpl, plcB, and prfA, were determined in L. monocytogenes isolates by PCR. These genes proved to play an essential role in the pathogenicity of L. monocytogenes. As a result, all isolates harbored at least nine of these genes. LIPI-1 (prfA, plcA, hly, mpl, actA, and plcB) genes were found in 55.2% (16/29) of the isolates, as well as the iap gene, which has an indirect role in their pathogenesis by codifying a product that is responsible for entering the host cell (Vázquez-Boland et al., 2001b; Iglesias et al., 2017). In addition, the inlA gene encodes a product that is responsible for the entry of the bacterium into the host cell, and the genes inlB, inlC, and inlJ are directly involved in the subsequent infection stages (Doumith et al., 2004; Iglesias et al., 2017). The L. monocytogenes strain isolated from the P. eryngii production plant contained inlA, inlC, and inlJ, whereas inlB was detected in 79.3% of isolates (23/29). However, the presence of either or both LIPI-1, inlA, inlB, inlC, and inlJ genes involved in the L. monocytogenes strain indicates that they had the ability to enter via the intraperitoneal route or through the use of contaminated food and cause infection. Furthermore, the mutations in inlA leading to a PMSC significantly reduce the invasion of the strain by human epithelial cells (Nightingale et al., 2005). However, only ten isolates from the sequencing of inlA genes had PMSC mutations, which means most of the strains (19/29, 65.5%) can produce full-length InlA, which is required for host cell invasion.

Regardless of the source, antimicrobial resistance within the isolates was significant. In this study, 17 clinically common antibiotics were tested for antimicrobial susceptibility in all L. monocytogenes strains. Compared with previous studies, the results of this study suggest that the overall incidence of antibiotic resistance in L. monocytogenes is relatively low (Pesavento et al., 2010; Rahimi et al., 2010; Camargo et al., 2015; Chen et al., 2018). Most L. monocytogenes isolates were sensitive to selected antibiotics. As the first-choice treatment for invasive listeriosis, only one isolate (3.5%, 1/29) and seven isolates (24.1%, 7/29) were resistant to ampicillin and penicillin. Besides, there was no resistance to the association of trimethoprim-sulfamethoxazole (an alternative treatment to invasive listeriosis). Erythromycin, a molecule recommended for the treatment of listeriosis in pregnant women (Charpentier and Courvalin, 1999), showed a mild level of resistance among the isolates (6.9% resistant and 6.9% intermediate resistance). L. monocytogenes is naturally susceptible to a wide range of antibiotics except for cephalosporin and fosfomycin (Hof et al., 1997). However, single- or multiple-resistant L. monocytogenes strains isolated from food, the environment, and clinics have been reported on a regular basis in recent years (Barbosa et al., 2013; Cufaoglu et al., 2021). Thus, as foodborne L. monocytogenes is the main cause of listeriosis, an antimicrobial susceptibility test for L. monocytogenes in food requires constant monitoring.

In summary, the results of this study suggested that the contamination of L. monocytogenes in P. eryngii (king oyster mushroom) production plants is most likely caused by mycelium stimulation machines, with low temperatures during growth being the most likely cause. In addition, the persistence of specific L. monocytogenes isolates (such as ST121 and ST3) may assist with contamination. Most isolates belonged to molecular serotypes 4b-4d-4e and 1/2a-3a, which are associated with human listeriosis, suggesting that this pathogen may represent a potential danger to public health. Furthermore, the presence of either or both LIPI-1, inlA, inlB, inlC, and inlJ genes involved in the L. monocytogenes strain indicates that they had the ability to enter via the intraperitoneal route or using contaminated food and cause infection. Further study of inlA sequencing showed that most of the selected strains contained PMSC-lacking inlA gene sequences required for the encoding of the InlA factor and bacterial invasion of the host cell. Although the isolates we have found were not highly resistant, antimicrobial susceptibility tests for L. monocytogenes in food require constant monitoring. Our study reflects the potential risk of L. monocytogenes contamination in P. eryngii production plants. However, effective Listeria monitoring programs will allow for the improved development of Listeria control measures to minimize cross-contamination in processing P. eryngii.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

SW and JX conceived and designed the experiments and analyzed the data. JX and ML performed the experiments. HH and YP contributed to reagents, materials, and analysis tools. SW, JX, and ZX contributed to the writing of the manuscript. All authors contributed to the article and approved the submitted version.