Development of Listeria monocytogenes detection technique in mushroom based on real-time quantitative PCR through improvement of enrichment medium

Abstract

The objective of this study was to develop precise and rapid detection methods for Listeria monocytogenes in mushrooms when using real-time PCR. L. monocytogenes were enriched through the optimization of the enrichment broth. To determine the optimal supplement for bacterial growth, the various components were investigated. As a result, LEB with 2×ferric citrate (LEB+2FC) was shown the most effective on promoting the L. monocytogenes growth in mushrooms. L. monocytogenes was inoculated in King Oyster mushroom and Golden needle mushroom, and the samples were enriched. Consequently, L. monocytogenes in King Oyster mushroom was detected by 100% (8/8) using real-time PCR when enriched in LEB+2FC for 9 h. In Golden needle mushroom, the pathogen was detected (100%; 8/8) when enriched in LEB+2FC for 3 h. These results indicate that L. monocytogenes can be detected quickly to prevent Listeria-related foodborne illness from the consumption of mushrooms with enrichment using LEB+2FC and real-time PCR analysis.

Introduction

Listeria monocytogenes, a member of the genus Listeria, naturally occurs in agricultural environments (Jeyaletchumi et al., 2012). Although L. monocytogenes infection is caused by the intake of ready-to-eat products such as dairy products, ham, frankfurthers, etc (Kim et al., 2008). Outbreaks of L. monocytogenes infections associated with fresh produce have been reported worldwide (Meldrum et al., 2009). Additionally, many studies have detected L. monocytogenes in fresh produce samples and even in some minimally processed vegetables. Thus, L. monocytogenes may contaminate fresh produce if presented in the cultivation environment (soil and water) (Zhu et al., 2017). Recently, 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; Wu et al., 2015). In the U.S., the outbreak caused by L. monocytogenes in March 2020 was reported due to the consumption of Golden needle mushrooms imported from the Republic of Korea. As a result, 36 illnesses, 31 hospitalizations, and 4 deaths occurred in 16 states because of this outbreakⁱ⁾. L. monocytogenes can survive and grow at low temperatures on packaged fresh-cut vegetables, and they can be contaminated in mushrooms that are collected from farms and distributed at low temperatures (Farber et al., 1998).

The standard microbiological methods, routinely adopted for the isolation of L. monocytogenes in foodstuffs or other materials, usually require two enrichment steps in liquid media and further isolation on a solid selective medium. Suspect colonies have subsequently to be confirmed through phenotypical characteristics (Barocci et al., 2008). Those conventional methods for the detection of L. monocytogenes in foods are labour-intensive and time consuming (Cox et al., 1998). Detection of the pathogen in fresh agricultural products using the selective growth medium is not appropriate because it takes 5 to 8 days (Oravcova et al., 2008). Even though this method detects a low concentration of L. monocytogenes in mushrooms, the mushroom already consumed when the positive result comes out. The low level of L. monocytogenes can multiply during the transport overseas or domestic distribution, which can cause foodborne illness.

Many molecular-based methods, including polymerase chain reaction (PCR), have been developed to overcome the limitations of conventional detection methods (Brauns et al., 1991). Studies have clearly shown that PCR-based methods are rapid, highly specific, and capable of detecting many pathogens (Hwang et al., 2010). PCR is a three-step cyclic in vitro procedure based on the ability of DNA polymerase to copy a strand of DNA (Malorny et al., 2009). However, this PCR approach requires analysis of the amplified DNA in an agarose gel or by DNA-DNA hybridization to confirm the results, which again is time-consuming and laborious (Panicker et al., 2004). SYBR Green (SG) is widely used in real-time quantitative PCR (qPCR) as an intercalating dye (Wittwer et al., 1997). Binding of SG to double-stranded DNA is non-specific and additional testing, such as DNA melting curve analysis, is required to confirm the generation of a specific amplicon. The use of melt curve analysis eliminates the necessity for agarose gel electrophoresis because the melting temperature (T_m) of the specific amplicon is analogous to the detection of an electrophoretic band (Giglio et al., 2003; Simpson et al., 2000). qPCR is characterized by high sensitivity, high reproducibility, and a rapid detection method. Another advantage is the decreased risk of cross-contamination due to the avoidance of further post-PCR processing, compared to conventional PCR (Hein et al., 2001). Pre-enrichment procedures are still necessary to ensure the detection of low numbers of viable L. monocytogenes in foods (Norton, 2002; O'Grady et al., 2008). To reach the detectable level of L. monocytogenes in qPCR analysis, the bacteria should be enriched through the optimization of the enrichment broth.

Therefore, this study developed a rapid detection method for L. monocytogenes in mushrooms using real-time PCR through the improvement of enrichment media.

Materials and Methods

Preparation of bacterial strain    L. monocytogenes strains (L. monocytogenes BL5-1, L. monocytogenes D2L3-1, L. monocytogenes HL1-1, L. monocytogenes IL1-1, and L. monocytogenes PL1-1) were isolated from Golden needle mushroom (Flammulina velutipes), and each strain was cultured in 10 mL tryptic soy broth with 0.6% yeast extract (TSBYE, Becton, Dickinson and Company, NJ, USA). One-hundred microliter aliquots were transferred to fresh 10 mL TSBYE, followed by incubation at 30 °C for 24 h. The cultures of the L. monocytogenes strains were mixed and centrifuged at 1 912 × g at 4 °C for 15 min. The pellets were washed twice with the same volume of phosphate buffered saline (PBS; 0.2 g KH₂PO₄, 1.5 g Na₂HPO₄, 8.0 g NaCl, and 0.2 g KCl in 1 L distilled H₂O [pH 7.4]). The suspension was diluted with PBS to obtain the appropriate bacterial level for each experiment.

Evaluation of modified enrichment broth    The original media components for Listeria Enrichment Broth (LEB; Becton, Dickinson and Company, USA) are shown in Table 1 to understand what components were adjusted. The components of LEB were changed with other materials or their concentrations for modified LEB. To determine the optimal ingredients for bacterial growth, the various components including carbon sources, nitrogen sources, sodium chloride, and selective agents were investigated (Table 2). The modified LEB was prepared by adding components, replacing components, or changing concentrations. LEB and modified LEB (9.9 mL) were placed into the sterilized tubes and inoculated with L. monocytogenes inoculum (0.1 mL), followed by incubating at 30 °C for 0, 3, 6, and 9 h. Each enriched broth was plated onto tryptic soy agar with 0.6% yeast extract (TSAYE, Becton, Dickinson and Company) to quantify L. monocytogenes. The plates were incubated at 30 °C for 24 h, and the colonies were manually counted.

Table 1. Composition of original Listeria enrichment broth medium

Medium components	Concentration (w/v%)
Tryptone	1.7
Soytone	0.3
Glucose	0.25
Sodium chloride	0.5
Dipotassium phosphate	0.25
Yeast extract	0.6
Cycloheximide	0.005
Acriflavin HCl	0.0015
Nalidixic Acid	0.004

Table 2. Components and concentrations used for modified Listeria enrichment broth

Medium components		Concentration (w/v%)
Carbon source	Glucose	0.15, 0.25
	Sucrose	0.15, 0.25
	Galactose	0.15, 0.25
Nitrogen source	Yeast extract	0.6, 1.2
	Beef extract	0.6, 1.2
	Yeast extract + beef extract	0.6 + 0.6
Sodium source	Sodium chloride	0.5, 1, 2, 3
Selective agent	Acriflavine HCl	0, 0.0015
	Lithium chloride	0.3
	Ferric citrate	0.05, 0.1

Evaluation and application of the modified LEB in mushrooms    King Oyster mushroom (Pleurotus eryngii) and Golden needle mushroom were purchased from a supermarket in Seoul, S. Korea, and they were placed aseptically into separate filter bags (3M, St. Paul, MN, USA) by 20 g. The sample with the lowest contamination of aerobic plate counts and L. monocytogenes was selected through the contamination level investigation. L. monocytogenes (0.1 mL) was inoculated onto the surface of the samples to obtain 2 Log CFU/g, and the samples were homogenized for 1 min and incubated at 30 °C for up to 11 h. Among the tested media, the most effective media (40 mL) was added after 20 g of the mushrooms were placed into the filter bag. They were homogenized for 1 min and incubated at 30 °C for 0, 3, 6, and 9 h for Golden needle mushroom and 0, 6, 9, and 11 h for King Oyster mushroom. The enriched samples were plated onto PALCAM (Oxoid Ltd) to quantify L. monocytogenes. The plates were incubated at 30 °C for 48 h, and the colonies were enumerated.

DNA extraction    DNA extraction was performed using DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. Three-milliliter aliquots of inocula and enriched samples were centrifuged at 5 264 × g at 4 °C for 10 min, and supernatants were discarded. Cell pellets were resuspended in 180 µL enzymatic lysis buffer and incubated at 37 °C for 30 min, and 25 µL proteinase K and 200 µL Buffer AL were added. The mixture was incubated at 56 °C for 30 min. Two hundred microliter of ethanol (96–100%) was added to the sample, and it was mixed thoroughly by vortexing. The mixture was flow through the DNeasy Mini spin column placed in a 2-mL collection tube. The collection tube was centrifuged at 5 989 × g for 1 min. The flow-through and collection tube were discarded. Five hundred microliters of Buffer AW1 and AW2 were added to DNeasy Mini spin column and centrifuged for 1 min at 5 989×g. Flow-through and collection tube were discarded and the DNeasy Mini spin column was placed in a microcentrifuge tube, and 200 µL Buffer AE was added directly onto the DNeasy Mini spin column. After incubating it at room temperature for 1 min, and then centrifuging for 1 min at 5 989 × g to elute. The eluent was then used for qPCR analysis.

qPCR for detection of L. monocytogenes in mushrooms    For the qPCR analysis, Roter-Gene SYBR^® Green PCR Kit (Qiagen) and the primer pair were used for the detection of L. monocytogenes. The primer pair composed of iapF (5′-TGG GAT TGC GGT AAC AGC AT-3′) and iapR (5′-TTA TCA ACA CCA GCG CCA CT-3′) (Lee et al., 2020). The detection limit of the iap primers previously performed is >1 Log CFU/mL. The qPCR reaction mixture (25 µL) contained l2.5 µL of SYBR Green master mix, 2.5 µL of each primer, distilled water, and the DNA template. The qPCR condition was as follows. Initial denaturation at 95 °C for 90 s; 35 cycles of denaturation at 95 °C for 5 s, annealing at 56 °C for 10 s, and extension at 72 °C for 5 s.

Statistical analysis    The SAS^® (Version 9.4, SAS Institute Inc, Cary, NC, USA) was used for statistical analysis. The values were expressed as mean ± standard deviation and analyzed using a general linear model (GLM) procedure. Mean comparisons were performed by a pairwise t test at α = 0.05.

Results and Discussion

Evaluation of modified LEB    In carbon sources, L. monocytogenes cell counts in LEB were increased by 1.0 Log CFU/mL after 9 h of enrichment, starting at 1.9 Log CFU/mL. LEB replaced with 0.25% sucrose was found to be the most effective among the three carbon sources (glucose, sucrose, and galactose) to increase L. monocytogenes cell counts. Compared to LEB, L. monocytogenes cell counts in this media were higher (p < 0.05) by 0.6 Log CFU/mL (Fig 1). Similarly, Busch and Donnelly (1992) study showed that heat-damaged L. monocytogenes were enriched best in sucrose and glucose among the seven different carbon sources (glucose, sucrose, lactose, mannose, fructose, and galactose).

Fig. 1.

Growth difference of Listeria monocytogenes after 9 h of enrichment at 30 °C under various carbons. LEB: Listeria enrichment broth, Glu: Glucose, Suc: Sucrose, Gal: Galactose. ^a–c) Means with different superscript letters are significantly different (p < 0.05).

LEB added with 1.2% yeast extract was most effective among nitrogen sources by increasing 1.5 Log CFU/mL of L. monocytogenes cell counts after 9 h, compared to the initial bacterial count. The differences in the enriched cell counts between LEB+1.2% yeast extract and LEB (1.0 Log CFU/mL) was 0.5 Log CFU/mL, and the doubled concentration of yeast extract in the LEB made it higher (p < 0.05) in the bacterial cell counts, compared to LEB (0.6% yeast extract) (Fig 2). Seeliger (1986) reported that yeast extract was an excellent source of the vitamin B complex that L. monocytogenes need. In the case of sodium chloride, a significant difference occurred at 2.0% when comparing the growth rate difference with LEB (0.5% sodium chloride) concentration (Fig 3).

Fig. 2.

Growth difference of Listeria monocytogenes after 9 h of enrichment at 30 °C under various nitrogens. LEB: Listeria enrichment broth, YE: Yeast extract, BF: Beef extract. ^a–c) Means with different superscript letters are significantly different (p < 0.05).

Figure 3.

Growth difference of Listeria monocytogenes after 9 h of enrichment at 30 °C under various sodium chloride concentrations. LEB: Listeria enrichment broth. ^a–b) Means with different superscript letters are significantly different (p < 0.05).

To select a selective agent for the LEB, the acriflavine HCl-free medium, the lithium chloride-added medium, and ferric citrate-added medium were used. L. monocytogenes cell counts in LEB were increased by 1.5 Log CFU/mL after 9 h of enrichment, from the initial bacterial counts (2.3 Log CFU/mL; Fig 4A). The selective agents originally composed of LEB are acriflavine HCl, which inhibits Gram-positive bacteria, and nalidixic acid which inhibits the growth of Gram-negative bacteria. In the case of cycloheximide, it inhibits the growth of mold. According to Seeliger (1972), the combination of acriflavine HCl and nalidixic acid effectively inhibit Gram-negative bacteria and Streptococci without affecting L. monocytogenes growth. On the other hand, a study by Beumer et al. (1996) reported that the generation time of L. monocytogenes was increased with a higher concentration of acriflavine HCl. For these reasons, the inhibitory effect of acriflavine HCl for the L. monocytogenes growth was investigated by its presence. As a result, acriflavine HCl was not modified from the composition of LEB since there was no significant difference when it was removed. A study by McBride and Girard (1960) reported that lithium chloride was effective in the proliferation of L. monocytogenes in the presence of Gram-negative bacteria. Therefore, lithium chloride was selected as a selective agent to inhibit Enterococci growth and to increase L. monocytogenes cell counts. As a result, when compared with LEB, the growth rate of L. monocytogenes was not significantly different (Fig 4B).

Fig. 4.

Growth difference of Listeria monocytogenes between LEB and Acriflavine HCl-free LEB (A) and according to selective agents (B) after 9 h of enrichment at 30°C. LEB: Listeria enrichment broth, AF: Acriflavine HCL, LC: Lithium chloride, FC: Ferric citrate. ^a–b) Means with different superscript letters are significantly different (p < 0.05).

The growth rate of L. monocytogenes was significantly increased only when ferric citrate was added, compared to LEB. Compared to the initial bacterial counts, L. monocytogenes cell counts increased by 2.3 Log CFU/mL in ferric citrate-added medium after 9 h enrichment, which was higher by 0.7 Log CFU/mL than in LEB (Fig. 4B). To determine the optimum level of ferric citrate, 2 × ferric citrate was added, but there was no significant difference between its levels (Fig. 4B). Iron is important for the growth of L. monocytogenes. For many bacterial pathogens, acquisition of iron from host proteins is a prerequisite for growth during infection (Brown and Holden, 2002). A study by Sword (1996) showed that the addition of iron compounds to enrichment media promoted the growth of L. monocytogenes. This bacterium possesses at least two iron acquisition systems. It has been reported to bind ferrous ions with the receptors on the bacterial cell surface and absorb them into cells. L. monocytogenes rapidly reduced iron from transferrin or, in the case of ferric ions, to recognize citrate and act as a transporter of iron through citrate-induced systems (Hartford et al., 1993; Cowart and Foster, 1985; Adams et al., 1990). This mechanism may increase L. monocytogenes cell counts with the addition of ferric citrate in the enrichment media.

To evaluate the growth difference of L. monocytogenes in mushrooms among LEB according to FC concentrations, the mushroom samples were inoculated with L. monocytogenes and cultured at 30 °C for 0, 6, 9, 11, and 12 h. In LEB, L. monocytogenes was increased, from 0.9 Log CFU/g to 3.9 Log CFU/g after 12 h of incubation, with the increment of 3.0 Log CFU/g, and there was no significant difference between LEB and LEB+FC. When in LEB+2FC, the initial bacterial counts (1.3 Log CFU/g) were increased by 3.4 Log CFU/g, reaching 4.7 Log CFU/g after 12 h of enrichment. The bacterial counts after 11 h only in LEB+2FC were significantly different, compared to LEB (p < 0.05) (Fig. 5). Hence, the most effective enrichment medium when applied to mushrooms was LEB+2FC, and it was selected as the optimal enrichment medium for further qPCR analysis. The reason for this selection was that the effect of carbon and nitrogen sources on L. monocytogenes growth are shown different results in studies, and in the case of FC addition, 2FC was more effective than FC when applied to mushroom. Also, since the current commercial media (LEB) can be easily used only by adding FC, only 2FC was added to the LEB to improve it. The compositions of LEB+2FC were as follows; tryptone 17 g, soytone 3 g, glucose 2.5 g, sodium chloride 5 g, dipotassium phosphate 2.5 g, yeast extract 6 g, cycloheximide 0.05 g, acriflavin HCl 0.015 g, nalidixic acid 0.04 g, and ferric citrate 1 g in 1 L distilled H₂O.

Fig. 5.

Growth difference of Listeria monocytogenes in mushroom during storage at 30 °C when using LEB+ferric citrate and LEB+2×ferric citrate. LEB: Listeria enrichment broth. FC: Ferric citrate. 2FC: 2×Ferric citrate.

^*Means with star indications are significantly different (p < 0.05), compared to control (LEB).

Application of detection system for L. monocytogenes in mushrooms    To establish the L. monocytogenes detection system for King Oyster mushroom and Golden needle mushroom, the optimum conditions for detecting L. monocytogenes in mushrooms with the modified enrichment medium were investigated. As a result of initial viable bacterial counts of samples, aerobic plate counts presented a higher contamination level in Golden needle mushroom (5.0 Log CFU/g) than King Oyster mushroom (2.9 Log CFU/g). In the case of L. monocytogenes, both were not detected.

In the case of the King Oyster mushroom, L. monocytogenes in LEB+2FC increased by 2.9 Log CFU/g after 11 h of incubation, compared to 0 h, while there was the lower increment (2.4 Log CFU/g) in LEB, compared to LEB+2FC (Table 3). To find the optimum enrichment time of L. monocytogenes, the bacterial cell numbers and the results of qPCR analysis under 9 h-incubation times were also investigated. At 9 h of incubation, the number of the bacterial cells enriched with LEB (3.7±0.4) and LEB+2FC (4.2±0.4) was enough to be detected by qPCR, however, the number of the positive samples was lower in LEB than that in LEB+2FC. (Table 3). Specifically, after enrichment of 9 h, qPCR detection showed that the positive rate of LEB+2FC was 100% (8/8), whereas there was 50% of accuracy (4/8) when enriched with LEB (Table 3), which was an unstable result. Also, the melting curves were stably expressed in all eight samples in LEB+2FC. Therefore, when detecting L. monocytogenes contaminated in King Oyster mushroom, with qPCR, the sample should be enriched in LEB+2FC at 30°C for 9 h.

Table 3. Listeria monocytogenes cell counts and C_T values in King Oyster mushroom (Pleurotus eryngii) samples enriched at 30 °C

Enrichment time(h)	Culture-based method (Mean ± SD; Log CFU/g)		Real-time PCR (Mean ± SD)
	LEB	LEB + 2FC1)	LEB	LEB + 2FC	Positive samples
					LEB	LEB + 2FC
0	1.8 ± 0.4	1.9 ± 0.4	-2)	-	-	-
6	2.9 ± 0.5	3.3 ± 0.5	ND3)	31.78 ± 0.18	0/4	2/4
9	3.7 ± 0.4	4.2 ± 0.4	30.84 ± 0.33	30.17 ± 1.84	4/8	8/8
11	4.2 ± 0.5	4.8 ± 0.5	30.34 ± 3.29	29.58 ± 1.29	8/8	8/8

¹⁾  LEB + 2× ferric citrate

²⁾  Not tested

³⁾  Not detected

In Table 4, the results of qPCR analysis when applied to Golden needle mushroom after enrichment were presented. The bacterial cell numbers in samples using LEB+2FC was 1.5 Log CFU/g at 3 h of enrichment at 30°C, while there was little growth in LEB. At 9 h-incubation, L. monocytogenes in LEB was increased by 1.6 Log CFU/g, whereas there was an increment of 2.2 Log CFU/g in LEB+2FC. (Table 4). According to qPCR analysis, 100% (8/8) of the accuracy rate was shown in both media after enrichment for 3 h. In the case of Golden needle mushroom, it may require at least 3 h of enrichment using LEB+2FC broth for the stable detection of L. monocytogenes by qPCR.

Table 4. Listeria monocytogenes cell counts and C_T values in Golden needle mushroom (Flammulina velutipes) samples enriched at 30 °C

Enrichment time(h)	Culture-based method (Mean ± SD; Log CFU/g)		Real-time PCR (Mean ± SD)
	LEB	LEB + 2FC1)	LEB	LEB + 2FC	Positive samples
					LEB	LEB + 2FC
0	1.0 ± 0.3	1.0 ± 0.5	-2)	-	-	-
3	1.1 ± 0.4	1.5 ± 0.4	31.23 ± 0.89	30.77 ± 0.93	8/8	8/8
6	2.0 ± 0.3	2.4 ± 0.5	-	-	-	-
9	2.6 ± 0.3	3.2 ± 0.4	-	-	-	-

¹⁾  LEB + 2× ferric citrate

²⁾  Not tested

The qPCR is an enzymatic reaction and therefore sensitive to inhibitors. qPCR inhibitors can originate from the sample or may be introduced during sample processing, and the polyphenols may hamper the PCR (Schrader et al., 2012). The total polyphenol contents of King Oyster mushroom and Golden needle mushroom are 387 mg% and 3.17–3.50 mg%, respectively (Ahn et al., 2006). King Oyster mushroom have higher polyphenols than Golden needle mushroom. Large amounts of polyphenols may act as inhibitors, making lower detection efficacy in King Oyster mushroom compared to Golden needle mushroom.

In conclusion, LEB+2FC can reduce the time taking to culture enough to detect L. monocytogenes in mushrooms with qPCR and improve the positive rate. Therefore, using this method for L. monocytogenes detection in mushrooms, the pathogen can be detected quickly and accurately to prevent Listeria-related foodborne illness from the consumption of mushrooms.

Author Contributions    Conceptualization, Y.Y; Methodology, Y.S. and Y.L.; Formal Analysis, Y.C. and H.O.; Investigation, J.K., E.P. and W.K.; Resources, Y.K.; Data Curation, S.K.; Writing – Original Draft Preparation, W.K.; Writing – Review and Editing, S.L.; Visualization, Y.Y. and M.S.; Supervision, H.L.; Project Administration, J.H.; Funding Acquisition, S.L.

Funding and Acknowledgment    This work was supported by the “Cooperative Research Program for Agriculture Science and Technology Development” Rural Development Administration, Korea [Project No. PJ013536].

Conflict of interest    There are no conflicts of interest to declare.

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