Prevalence and contamination levels of listeria monocytogenes in ready-to-eat foods in Tokyo, Japan.

We surveyed prevalence and contamination levels of Listeria monocytogenes in ready-to-eat foods between 2000 and 2012 in Tokyo. L. monocytogenes was isolated from 52 (1.7%) out of 2,980 samples. Comparing the prevalence in the study period, 2.2% were positive in the former period (2000-2005) and 1.2% in the latter (2006-2012). Using the most probable number (MPN) technique, 32 samples were contaminated with fewer than 0.3 L. monocytogenes/g, 10 samples with 0.3-1.0/g and 4 samples with more than 1.0/g (the maximum was 2.3/g). The most common serovar was 1/2a, followed by 1/2b, 4b and 1/2c. We revealed that ready-to-eat foods in Tokyo were contaminated with L. monocytogenes, although the contamination levels were low.

Listeria monocytogenes is a food-borne pathogen. Although it causes mild, non-specific and influenza-like symptoms in healthy adults, it can cause invasive listeriosis culminating in sepsis and meningitis in the immunocompromised, and the elderly. When pregnant women are infected with this organism, it is particularly devastating for unborn or newly delivered babies [4].

Only one food-borne outbreak caused by L. monocytogenes has been reported in Japan, which was due to natural cheese in 2001 [10]. In the U.S.A., European countries and Australia, outbreaks were often reported due to dairy foods, meat products, deli meat, fish products and vegetables [4].

In 2007, the Codex Alimentarius Commission has presented the guidelines to control L. monocytogenes in foods [2], and microbiological criteria for L. monocytogenes were adopted in ready-to-eat (RTE) foods prepared for consumption without further heating required as an annex in the document in 2009 [3]. The Codex determined that samples should contain L.monocytogenes no more than 100 cfu/g for foods in which growth will not occur and that samples should show absence in 25 g for foods in which growth can occur. The Codex gave the following examples of factors that can control growth of L. monocytogenes in foods: pH below 4.4, water activity (Aw) <0.92, a combination of factors (pH, Aw) e.g., a combination of pH <5.0, Aw <0.92 and freezing.

In Japan, soft or semi-soft cheese and unheated meat products contaminated with L. monocytogenes have been prohibited for import or commercial use since 1993 [12]. These foods should have shown absence of this organism in 25 g. To follow the Codex microbiological criteria, the standard for soft/semi-soft and semi-hard cheese and unheated meat products was fixed at ≤100 cfu/g in 2014 [13, 14]. In the present study, we surveyed the prevalence and the extent of contamination of this organism and other Listeria spp. in many types of RTE foods in Tokyo, Japan. As preservatives have been developed to control Listeria spp. in RTE foods recently, the period of collection was divided into the former and the latter to compare the prevalence between the two periods. And, pH and Aw were determined for the RTE foods found to be contaminated with L. monocytogenes, to determine whether growth of this organism could occur.

A total of 2,980 samples were obtained from 2000 to 2012 from retail shops and food-processing plants in metropolitan Tokyo and surrounding area. These included 626 dairy products, 1,491 meat and meat products, 718 seafood samples and 145 pickled vegetable products (Table 1). The period of collection was divided into “former” (from 2000–2005) and “latter” (from 2006–2012). The total number of samples collected in the former and the latter period was 1,710 and 1,270, respectively.

Table 1.

Prevalence of Listeria spp. in ready-to-eat foods in Tokyo (2000–2012)

	Samples	Period	No. of samples examined	No. of positive samples (%)
				L. monocytogenes	L. innocua	L. seeligeri	L. welshimeri	L. grayi	Listeria spp.
Dairy products
	Natural cheese (Soft/semi-soft)	2000–2005	127	1 (0.8)	4 (3.1)				5 (3.9)
		2006–2012	161		1 (0.6)				1 (0.6)
		Subtotal	288	1 (0.3)	5 (1.7)				6 (2.1)
	Natural cheese (Hard/semi-hard)	2000–2005	217	1 (0.5)					1 (0.5)
		2006–2012	73						0 (0)
		Subtotal	290	1 (0.3)					1 (0.3)
	Dehydrated powdered infant formula	2000–2012	39						0 (0)
	Butter	2000–2012	8						0 (0)
	Cream	2000–2012	1						0 (0)
Total			626	2 (0.3)	5 (0.8)				7 (1.1)
Ready-to-eat raw meats and meat products
	Ready-to-eat raw meats	2000–2005	83	3 (3.6)	8 (9.6)		5 (6)		12 (14.5)
		2006–2012	13						0 (0)
		Subtotal	96	3 (3.1)	8 (8.3)		5 (5.2)		12 (12.5)
	Unheated meat products	2000–2005	85	13 (15.3)	6 (7.1)		2 (2.4)		19 (22.4)
		2006–2012	43	1 (2.3)	2 (4.7)		2 (4.7)	1 (2.3)	3 (7)
		Subtotal	128	14 (10.9)	8 (6.3)		4 (3.1)	1 (0.8)	22 (17.2)
	Specifically heated meat products	2000–2005	27				3 (11.1)		3 (11.1)
		2006–2012	10						0 (0)
		Subtotal	37				3 (8.1)		3 (8.1)
	Heat treated meat products (packaged after being heat treated)	2000–2005	474	2 (0.4)	4 (0.8)		1 (0.2)		5 (1.1)
		2006–2012	617	7 (1.1)	2 (0.3)		2 (0.3)		10 (1.6)
		Subtotal	1091	9 (0.8)	6 (0.5)		3 (0.3)		15 (1.4)
	Heated meat products (heat treated after being packaged)	2000–2012	57						0 (0)
	Dried meat products	2000–2012	40						0 (0)
	Cooked meats	2000–2005	37				1 (2.7)		1 (2.7)
		2006–2012	5						0 (0)
	Subtotal	42				1 (2.4)		1 (2.4)
Total			1491	26 (1.7)	22 (1.5)		16 (1.1)	1 (0.1)	53 (3.6)
Ready-to-eat seafood samples
	Fresh fish	2000–2005	38	1 (2.6)	3 (7.9)				4 (10.5)
		2006–2012	48	4 (8.3)	7 (14.6)	4 (8.3)		1 (2.1)	11 (22.9)
		Subtotal	86	5 (5.8)	10 (11.6)	4 (4.7)		1 (1.2)	15 (17.4)
	Shellfish	2000–2005	111	1 (0.9)	1 (0.9)	1			3 (2.7)
		2006–2012	49						0 (0)
		Subtotal	160	1 (0.6)	1 (0.6)	1 (0.6)			3 (1.9)
	Boiled seafood	2000–2012	19						0 (0)
	Roe	2000–2005	79	5 (6.3)	13 (16.5)	1 (1.3)			17 (21.5)
		2006–2012	45	1 (2.2)	4 (8.9)			1 (2.2)	6 (13.3)
		Subtotal	124	6 (4.8)	17 (13.7)	1 (0.8)		1 (0.8)	23 (18.5)
	Delicatessen of seafood	2000–2005	108	2 (1.9)	10 (9.3)	1 (0.9)			12 (11.1)
		2006–2012	90	2 (2.2)	1 (1.1)			1 (1.1)	4 (4.4)
		Subtotal	198	4 (2)	11 (5.6)	1 (0.5)		1 (0.5)	16 (8.1)
	Dried seafood	2000–2005	131	5 (3.8)	9 (6.9)				12 (9.2)
Total			718	21 (2.9)	48 (6.7)	7 (1)		3 (0.4)	69 (9.6)
Pickled vegetables
	Pickled vegetables	2000–2005	145	3 (2.1)					3 (2.1)

For each sample, one-step enrichment method and/or two-step enrichment method was adopted. One-step enrichment method was performed as follows. Aseptically composited 25-g portions of samples were homogenized with 225 ml of enrichment medium and then incubated at 30°C for 48 hr. For enrichment, EB broth (Merck, Darmstadt, Germany) was used for dairy products, and UVM broth (Merck) was used for all other samples. Two-step enrichment method was performed as follows, 25-g portions of test samples were added to 225 ml of primary enrichment medium; half-Fraser broth (Merck) and incubated at 30°C for 24 hr. Primary enrichment cultures (0.1 ml) were inoculated into 10 ml of secondary enrichment medium; Fraser broth (Merck) and incubated at 37°C for 48 hr. Each enrichment culture was plated out on PALCAM agar and incubated at 30°C for 48 hr. The cultures of some samples were plated out on ALOA (Merck) or CHROMagar Listeria (CHROMagar, Paris, France) plates and incubated at 37°C for 24–48 hr. From each plate, colonies presumptively identified as Listeria spp. were streaked onto trypticase soy agar (Eiken Chemical, Tokyo, Japan) and incubated at 30°C for 24 hr to obtain pure culture. Confirmation that isolates were Listeria spp. was carried out by performing the Henry illumination test, catalase reaction, motility test, CAMP test, and assessing carbohydrate utilization of rhamnose, xylose and mannitols based on the use of ISO11290-1 method [8].

Quantitative determination was conducted using the most probable number (MPN) method with 3 tubes. Briefly, 25-g portions of test samples were added to 225 ml of culture medium and homogenized. Then, 10 ml of the homogenate was added to each of the 3 empty tubes, and 1 ml and 0.1 ml of the homogenate to each of 3 tubes of 10 ml culture medium, which were incubated at 30°C for 48 hr. For the culture medium, EB broth was used for dairy products, and UVM broth was used for all other samples. After incubation, each culture was plated out on PALCAM agar and incubated at 30°C for 48 hr. From each plate, colonies presumed to be Listeria spp. were checked for confirmation. MPN value was estimated on the basis of the number of positive tubes obtained from each set of dilution series.

Cultures identified as L. monocytogenes were serotyped using commercially available Listeria anti-sera (Denka Siken Co., Tokyo, Japan).

The pH of the samples was estimated using a pH meter by measuring the filtrate of samples with distilled water. The Aw of the samples was measured using a Decagon Model CX-2 (Decagon Devices, Inc., Pullman, WA, U.S.A.).

The prevalence of Listeria spp. in RTE foods is shown in Table 1. L. monocytogenes was isolated from 52 (1.7%) out of 2,980 samples. Comparing the prevalence of L. monocytogenes in the study period, 37 (2.2%) out of 1,710 samples were found positive in the former period, and 15 (1.2%) out of 1,270 samples in the latter.

A risk assessment report of L. monocytogenes in foods by the Food Safety Commission of Japan states that the percentage of retail foods contaminated with L. monocytogenes was 2.2% for imported natural cheeses, 0% for domestic natural cheese, 0% for other dairy goods, 3.89% for unheated meat products, 2.26% for fresh seafood, 7.19% for seafood products and 18.8% for pickled vegetables [6]. A previous risk assessment conducted by the FDA/USDA/CDC showed that the percentages for contaminated foods in the U.S.A. were 2.5% for cheese, 0.3% for dairy goods other than cheese, 3.0% for processed meat products, 7.0% for fresh seafood, 9.5% for seafood products and 2.1% for pickles [5]. Comparing prevalence of contamination among reports, the percentage of food contaminated with L. monocytogenes of natural cheese (0.3%) and pickled vegetables (2.1%) was low, and that of unheated meat products (10.9%) was high in our study.

The period of the survey (2000–2012) was divided into two parts, the former and the latter. The contamination of food products, such as unheated meat products and roe, tended to be reduced in the latter half of the study period. Recently, good hygienic practices [2] and use of preservatives, such as sodium benzoate, sodium propionate, potassium sorbate and nisin [19], have been introduced to limit contamination with Listeria spp. in many manufacturing facilities in Japan and overseas. As such, contamination with this pathogen may tend to be reduced.

In pickled vegetables, L. monocytogenes was detected in 3 (cucumber pickled in rice-bran paste, 2.1%) of 145 samples. It has been reported in Hokkaido where factory contamination with L. monocytogenes was suspected during the production of “asazuke”, Japanese light pickles [11]. In 2012, an outbreak of enterohemorrhagic Escherichia coli O157 due to consumption of pickled Chinese cabbage occurred in a number of districts [7]. Therefore, pickled vegetables should be applied general principles of food hygiene, and their sanitary facilities must be improved in the factory setting to prevent secondary contamination from the production line in the same way as dairy foods or meat products.

From the positive samples, 46 were assessed for numbers of L. monocytogenes by MPN. Thirty-two of these were contaminated with <0.3 L. monocytogenes/g, 10 with 0.3–1.0/g and 4 with >1.0/g (Table 2). The samples contaminated with >1.0/g were a semi-soft cheese type made in France (value, 1.6/g), 2 minced tuna samples (values, 2.1/g and 2.3/g) and “matsumaezuke,” which is a kind of pickle of squid and seaweeds (value, 1.5/g).

Table 2.

MPN value of L. monocytogenes in ready-to-eat foods (2000–2012)

Food samples	No. of samples examined	MPN value (/g)
		<0.3	0.3–1	>1
Natural cheese	2	1		1a)
Ready-to-eat raw meats	3	3
Unheated meat products	13	10	3
Heat treated meat products (packaged after being heat treated)	7	3	4
Fresh fish	5	2	1	2b)
Roe	5	5
Delicatessen of seafood	4	2	1	1c)
Dried seafood	4	4
Pickled vegetables	3	2	1
Total	46	32	10	4

a) Semi-soft cheese made in France, b) Minced tuna, c) “Matsumaezuke”, a kind of pickle of squid and seaweeds.

The pH and Aw of 11 samples out of L. monocytogenes-positive samples are shown in Table 3. Samples that were pH ≥4.4, Aw ≥0.92 and pH ≥5.0 + Aw ≥0.94 were 1 of 4 unheated meat products, 1 heat treated meat product (packaged after being heat treated), 4 fresh fish samples and 1 of 2 fish products (Table 3).

Table 3.

pH and Aw of ready-to-eat foods contaminated with L. monocytogenes

Type of foods	Samples	MPN value (/g)	pH	Aw
Unheated meat products	Salami sausage	<0.3	7.0	0.93
	Salami sausage	<0.3	6.4	0.83
	Raw ham	0.4	6.1	0.91
	Raw ham	<0.3	6.0	0.89
Heat treated meat products(packaged after being heat treated)	Sausage	0.36	6.4	0.98
Fresh fish	Minced tuna	0.92	5.8	0.99
	Minced tuna	<0.3	6.1	0.98
	Minced tuna	2.3	6.2	0.99
	Minced tuna	<0.3	6.9	0.98
Roe	“Tarako’’	<0.3	6.0	0.95
Delicatessen of seafood	Jellyfish with sea urchin eggs	<0.3	5.8	0.86

In this survey, it was almost less than 1/g, and the highest value observed was 2.3/g. However, in this study, L. monocytogenes was detected in foods where pH and Aw supported growth [3]. Although the MPN value of L. monocytogenes being low, it is possible for the organism to grow in those foods. It has been reported that generation times of L. monocytogenes are 36 hr at 4.4°C and 10 hr at 10°C [18]. Furthermore, despite the fact that raw RTE seafood in Japan has a short shelf life, cell numbers of L. monocytogenes in minced tuna and salmon roe increased rapidly under inappropriate storage temperatures of 10°C (from a MPN of 10° to 101/g to a MPN of 103 to 104/g over a course of 2 days at 10°C) [15]. The Codex guideline prescribes keeping the temperature at less than 6°C for production and transportation [2]. As the Codex, raw RTE seafood should be kept at less than 6°C in Japan.

Serovars of 63 isolates identified as L. monocytogenes isolated from RTE foods included 1/2a, 30 strains (47.6%); 1/2b, 13 strains (20.6%); 4b, 9 strains (14.3%); 1/2c, 7 strains (11.1%); 3b, 2 strains (3.2%); 3a and 3c, 1 strain (1.6%), respectively (Table 4). In the report of Food Safety Commission of Japan, that was 1/2a (52.2%), followed by 1/2c (17.2%), 1/2b (15.3%) and 4b (10.2%) [6]. These findings were similar. On the other hand, the most common serotype from case of listeriosis in Japan during 1958–2001 was 4b (59.9%), followed by 1/2b (26.4%) and 1/2a (5.8%) [6]. The most common serotype was different between isolates obtained from humans and food, probably due to differences in pathogenicity. It has been reported that L. monocytogenes can be attenuated, depending on the mutation of virulence-associated genes, such as prfA, inlA, inlB and plcA in lineage II [17]. The exhibit of mutations result in a premature stop codon in inlA is different for each serotype, 19.1% in 1/2a, 14.3% in 1/2b, 100% in 1/2c and 0% in 4b [9]. Otherwise, after 2000, serotype 4b was still commonly reported in cases in the U.S.A. [1], but serotype 1/2a showed an increasing trend in Europe [16]. The common serotype in the outbreak was 4b before the year 2000. However, serotype 1/2a increased after 2000 [6]. It is possible that the prevalent serotype of L. monocytogenes is changing recently.

Table 4.

Serotypes of L. monocytogenes isolated from ready-to-eat foods (2000–2012)

Food samples	No. of isolates of each serotypes (%)
	1/2a	1/2b	1/2c	4b	3a	3b	3c	Total
Natural cheese	1 (50)	1 (50)						2
Ready-to-eat raw meats and meat products	17 (51.5)	6 (18.2)	2 (6.1)	6 (18.2)		1 (3)	1 (3)	33
Ready-to-eat seafood samples	10 (40)	5 (20)	5 (20)	3 (12)	1 (4)	1 (4)		25
Pickled vegetables	2 (66.7)	1 (33.3)						3
Total	30 (47.6)	13 (20.6)	7 (11.1)	9 (14.3)	1 (1.6)	2 (3.2)	1 (1.6)	63

The RTE foods marketed in Tokyo were contaminated with this organism, although the contamination levels were low. There are few incidents or outbreaks of L. monocytogenes in Japan, but Japan Nosocomial Infections Surveillance estimated that 200 listeriosis cases occur in a year [6]. Considering the high mortality rate this disease presents with, many of these are likely to have been fatal. In addition, listeriosis is largely attributable to foodborne transmission of the microorganism. It is important to continue surveys of prevalence and contamination levels of L. monocytogenes in RTE foods in Tokyo, Japan.