Prevalence and Populations of Listeria monocytogenes in Meat Products Retailed in Sao Paulo,Brazil

Abstract

This study evaluated the prevalence of the populations and serotypes of Listeria monocytogenes in 552 refrigerated samples of ground beef, chicken leg, hot dog, and pork sausage collected in supermarkets in the city of Sao Paulo, SP, Brazil, between May 2008 and July 2009. The supermarkets were selected after stratification by geographical region and by random draw. Tests for presence and enumeration of L. monocytogenes were based on ISO 11290-1:1996/Amd.1:2004 and ISO 11290-2:1998 methods, respectively. Listeria spp. were detected in 469 (85.0%) of the studied meat products. The most frequently isolated species was L. innocua (64.1%), followed by L. monocytogenes (48.7%), L. welshimeri (13.4%), L. seeligeri (7.1%), L. ivanovii (0.2%), and L. grayi subspecies murrayi (0.2%). L. monocytogenes was detected in 269 (48.7%) samples, with highest prevalence in ground beef (59.4%) followed by chicken legs (58.0%), pork sausages (39.8%), and hot dogs (37.7%). The populations were <10² colony-forming units/g in the majority of samples (62.5%). Prevalence of serotypes varied according to the type of meat product. These data are relevant for estimating the risks of listeriosis associated with consumption of meat products in Sao Paulo, and for establishing science-based intervention strategies aimed at reducing these risks, especially for pregnant women and immunocompromised individuals.

Introduction

L isteria monocytogenes is a bacterium widely distributed in nature that can cause listeriosis, a serious foodborne infection, with low morbidity but high mortality (Rocourt et al., 2000). The disease affects mainly pregnant women, newborn, the elderly, and individuals with acquired or induced immunosuppression, such as those carrying the human immunodeficiency virus, transplant recipients, and those with diabetes or under chemotherapy (Swaminathan et al., 2007; Pouillot et al., 2012). Listeriosis in humans is characterized by infections of the central nervous system (meningitis, encephalitis, and meningoencephalitis), primary bacteremia and septicemia (Vazquez-Boland et al., 2001; Allerberger et al., 2010), and in healthy people the disease can manifest like an influenza or a febrile gastroenteritis (Hof, 2001; Vivant et al., 2013). The infectious dose of L. monocytogenes is not established, but can vary from 10² to 10⁹ colony-forming units (CFU)/g depending on the immunological status of the host (Jemmi and Stephan, 2006).

Many foods have been implicated in cases of listeriosis, such as vegetables, raw milk, meat products and their derivatives, and ready-to-eat foods (Jemmi and Stephan, 2006; Swaminathan et al., 2007; Cartwright et al., 2013; CDC, 2013). L. monocytogenes can be introduced in meat products during processing, and the degree of contamination may depend on the extent of cross-contamination, hygienic measures by food handlers, and the processing parameters (Zhu et al., 2012). Furthermore, since Listeria are able to grow at refrigeration temperatures, conditions at the retail level, during transport, and home storage may allow pathogen growth to hazardous levels prior to consumption.

Of the 13 L. monocytogenes serotypes identified to date, three (1/2a, 1/2b, and 4b) are frequently associated with human clinical cases of listeriosis. The serotype 4b is involved in almost all outbreaks (Fox et al., 2012; Pontello et al., 2012; Cartwright et al., 2013), suggesting that strains belonging to this serotype are better adapted to the mammalian host tissues than strains from serotype 1/2 (Vazquez-Boland et al., 2001).

In most countries, including Brazil, risk assessments are difficult to undertake due to numerous knowledge gaps, especially the scarcity of available epidemiological data on prevalence and characteristics of foodborne illnesses in the country and the lack of quantitative data regarding pathogenic microorganisms in the food chain.

In Brazil, cases of human listeriosis are under-reported and there is no evidence of foodborne transmission (Martins et al., 2010; Blum-Menezes et al., 2013). However, several studies have reported the presence of L. monocytogenes in meat products, such as pork sausage (Miyasaki et al., 2009; Monteiro et al., 2013), hot dog (Pettinati et al., 2006), and ground beef (Monteiro et al., 2013). Quantification of L. monocytogenes in meat products was done in only a small number of these studies (Miyasaki et al., 2009; Martins and Germano, 2011).

This work was performed with the purpose of generating qualitative and quantitative data on L. monocytogenes in meat products marketed in Sao Paulo that could be used for future risk assessments.

Materials and Methods

Samples

The study was carried out with 552 samples of meat products (138 of hot dog, 138 of pork sausage, 138 of ground raw beef, and 138 of raw chicken leg) purchased in 138 supermarkets located in different geographic regions (central, west, east, north and south) of the city of Sao Paulo, SP, Brazil. The supermarkets were selected after stratification by geographical region and by random draw, covering 80.2% of the city districts (IBGE, 2009). The samples were collected directly from the vending shelves, transported to the laboratory in isothermal boxes, and kept under refrigeration until tested (a maximum of 18 hours).

Detection and enumeration of L. monocytogenes

Twenty-five grams of each sample were analyzed for the detection (ISO 11290-1:1996/Amd. 1:2004) and enumeration (ISO 11290-2:1998) of pathogen. Briefly, selective enrichments Half-Fraser (HF) and Fraser (Oxoid, Hampshire, UK) were used for the investigation and streaked on Palcam agar plates and agar Listeria according to Ottaviani and Agosti (ALOA) plates. For the enumeration, 1-mL and 0.1-mL aliquots of 1:10 and 1:100 dilutions of the HF broth were plated on ALOA and Palcam plates. Following incubation of ALOA and Palcam plates, three to five characteristic colonies of Listeria in each plate were isolated on tryptic soy agar plates containing yeast extract. Colonies were submitted to Gram staining and tested for production of catalase, β-hemolysis, and fermentation of carbohydrates (xylose, mannitol, and rhamnose). Results were expressed as presence or absence of L. monocytogenes per 25 g (prevalence) or CFU/g (enumeration).

Serotyping of L. monocytogenes

The molecular serotyping was performed according to Doumith et al. (2004), with modifications, using multiplex–polymerase chain reaction (PCR) for the genes prs, ORF2819, ORF2110, lmo0737, and lmo1118. The procedure included a denaturation cycle at 94°C for 3 min, followed by 35 cycles of 40 s at 94°C, 75 s at 55°C, and 75 s at 72°C, and a final extension at 72°C for 7 min. At least two isolates of L. monocytogenes from each positive meat product were submitted to molecular serotyping. The DNA was extracted from cells using Wizard^® Genomic DNA Purification System (Promega, Madison, WI), following the manufacturer's instructions.

Statistical analysis

To evaluate the association of positive results for L. monocytogenes and the geographical region where the samples were collected, the chi-square test, according to Siegel (1975), was used. The significance level was 5%.

Results and Discussion

Listeria spp. were detected in 85.0% (469/552) of the studied meat products (Table 1). The most frequently isolated species was L. innocua (64.1%), followed by L. monocytogenes (48.7%), L. welshimeri (13.4%), L. seeligeri (7.1%), and L. ivanovii (0.4%). L. grayi subspecies murrayi was detected in only one sample of beef sausage (0.2%) (Table 2). These results confirm previously reported data in literature that indicate that L. innocua is the most common species in foods (Chen et al., 2009; Derra et al., 2013). A total of 228 (41.3%) samples presented two or more Listeria species. The presence of multiple Listeria species in a single sample may be related to the ubiquitous nature of these microorganisms and cross-contamination at the retail level resulting from storage conditions and the handling of these products. In some supermarkets, different types of meat are stored and handled in the same place and by the same person, favoring the contamination. Furthermore, inappropriate cleaning of the environment and utensils such as knives and meat grinders, coupled with capacity of Listeria spp. to form biofilms and multiply at refrigeration temperature, contributes to persistence and dissemination of the pathogen.

Table 1.

Distribution of Listeria Species in Meat Product Samples Marketed in Sao Paulo, Brazil

	No. positive samples
Species of Listeria	Hot dog	Pork sausage	Ground beef	Chicken leg	Total (%)
Lm	22	14	15	20	71 (12.9)
Lm+Lin	20	24	57	47	148 (26.8)
Lm+Ls	4	0	0	1	5 (0.9)
Lm+Lw	1	5	0	2	8 (1.4)
Lm+Liv	0	0	0	1	1 (0.2)
Lm+Lin+Ls	1	3	3	1	8 (1.4)
Lm+Lin+Lw	2	8	4	8	22 (4.0)
Lm+Lw+Ls	1	1	0	0	2 (0.4)
Lm+Lin+Ls+Lw	0	0	3	0	3 (0.5)
Lm+Lin+Lw+Lg	1	0	0	0	1 (0.2)
Lin	37	26	42	39	144 (26.1)
Lin+Ls	3	4	3	1	11 (2.0)
Lin+Lw	0	10	2	4	16 (2.9)
Lin+Lw+Ls	0	1	0	0	1 (0.2)
Ls	5	0	1	1	7 (1.3)
Lw	3	9	0	7	19 (3.4)
Lw+Ls	0	1	0	1	2 (0.4)
Total (%)	100 (72.5)	106 (76.8)	130 (94.2)	133 (96.4)	469 (85.0)

Lm, L. monocytogenes; Lin, L. innocua; Ls, L. seeligeri; Liv, L. ivanovii; Lw, L. welshimeri; Lg, L. grayi.

Table 2.

Prevalence of Listeria Species in 552 Meat Product Samples Marketed in Sao Paulo, Brazil

	No. positive samples (%)
Listeria species	Hot dog	Pork sausage	Ground beef	Chicken leg	Total
L. monocytogenes	52 (37.7)	55 (39.8)	82 (59.4)	80 (58.0)	269 (48.7)
L. innocua	64 (46.4)	76 (55.1)	114 (82.6)	100 (72.5)	354 (64.1)
L. welshimeri	8 (5.8)	35 (25.4)	9 (6.5)	22 (15.9)	74 (13.4)
L. seeligeri	14 (10.1)	10 (7.2)	10 (7.2)	5 (3.6)	39 (7.1)
L. ivanovii	0 (0)	0 (0)	0 (0)	1 (0.7)	1 (0.2)
L. grayi	1 (0.7)	0 (0)	0 (0)	0 (0)	1 (0.2)

The overall prevalence of L. monocytogenes in meat products was 48.7% (269/552), being detected in 82 (59.4%) samples of ground beef, 80 (58.0%) of chicken leg, 55 (39.8%) of pork sausage, and 52 (37.7%) of hot dog (Table 2). These numbers are higher than those reported in most studies conducted in Brazil (Pettinati et al., 2006; Barros et al., 2007; Miyasaki et al., 2009; Monteiro et al., 2013). The differences in the results among these studies may be related to the sampling techniques, meat production and processing, region of sampling, and laboratory methodologies.

There is a wide variation in the occurrence of L. monocytogenes in meat products in different countries. In raw chicken, the prevalence can vary from 15.7% in China (Zhang et al., 2013) to 70% in Estonia (Praakle-Amin et al., 2006), in ground beef from 5% in the United States (Pao et al., 2009) to 29% in Ireland (Khen et al., 2014), and in samples of pork sausages from 3.7% in Spain (Cabedo et al., 2008) to 42% in Italy (Meloni et al., 2009).

No association between the presence of L. monocytogenes in meat products and the geographical regions of sample collection could be obtained (p>0.05). However, the prevalence of L. monocytogenes in ground beef samples obtained in the northern region of the city of Sao Paulo (>70%) was significantly higher (p<0.05) than in other regions. Differences in the cleaning frequency and efficiency of grinding equipment, grinding time, and/or improper product storage in these supermarkets may have contributed to the observed results.

Since risk assessments require more complete data to estimate the impact of L. monocytogenes on consumer health, we also determined the level of contamination in the meat product samples. In most samples (62.5%), the counts were below 10² CFU/g. In hot dogs, the values ranged from <10 to 1.9×10² CFU/g, in pork sausage varied from <10 to 5.6×10² CFU/g, in chicken leg from <10 to 8.9×10² CFU/g, while in ground beef the values were from <10 to 6.3×10³ CFU/g (Table 3). These results corroborate those reported in other studies with similar meat products in Brazil (Miyazaki et al., 2009), Poland (Modzelewska-Kapituła et al., 2014), and Ireland (Khen et al., 2014).

Table 3.

Distribution of the Meat Product Samples According to the Counts of Listeria monocytogenes

	Counts of L. monocytogenes (CFU/g)
Type of meat	<10	10 to 10²	>10² to ≤10³	>10³
Hot dog	129	7	2	0
Pork sausage	127	9	2	0
Ground beef	82	34	17	5
Chicken leg	119	14	5	0
Total	457 (82.8)	64 (11.6)	26 (4.7)	5 (0.9)

CFU, colony-forming units.

Despite the low counts of L. monocytogenes in the tested meat products, the pathogen is psychrotrophic and can grow during storage under refrigeration. In hot dog samples, L. monocytogenes can increase 1.5 logs during storage for 7 days at 7°C (Simpson Beauchamp et al., 2010). The consequences for human health can be serious if these products are ingested without a thermal treatment or are allowed to cross-contaminate other food products (Nørrung, 2000; Thevenot et al., 2006).

Molecular serotyping of 442 L. monocytogenes strains isolated from 95 samples of ground beef (143 strains), 93 samples of chicken leg (134 strains), 59 samples of pork sausage (83 strains), and 55 samples of hot dog (82 strains) indicated that they were equally distributed in the four serotype groups: 28.7% belonged to Group 1 (1/2a and 3a), 21.0% to Group 2 (1/2c and 3c), 17.0% to Group 3 (1/2b, 3b, and 7), and 13.8% to group 4 (4b, 4d, and 4e) (Table 4). In three strains (0.7%) only the prs gene was amplified, indicating that these strains probably belong to serotype 4a, 4c, or another species of Listeria.

Table 4.

Prevalence of Listeria monocytogenes Serotypes According to the Type of Meat Product

	Molecular serotyping
Type of meat	Group 1 (1/2a, 3a)	Group 2 (1/2c, 3c)	Group 3 (1/2b, 3b, 7)	Group 4 (4b, 4d, 4e)	4a or 4c	Non- typeable	Total
Hot dog	19	8	32	15	0	8	82
Pork sausage	41	18	11	10	1	2	83
Ground beef	7	38	22	22	1	53	143
Chicken leg	60	29	10	14	1	20	134
Total (%)	127 (28.7)	93 (21.0)	75 (17.0)	61 (13.8)	3 (0.7)	83 (18.8)	442 (100)

In 18.8% of the strains, the serotyping by multiplex-PCR resulted in a novel atypical profile named IVb-v1 by Leclercq et al. (2011), where four DNA fragments were amplified, one of which is characteristic of the genus Listeria (prs), two are characteristic of Group 4 (ORF2819 and ORF2110), and one is characteristic of Group 1 (Imo737). The presence of these four fragments was confirmed by PCR for each of the genes. The PCR profile was the same as those described in Brazil and in other regions (including France, Algeria, Switzerland, United States, and Australia), which serotyped the isolates by conventional serology and found those that belong to serotype 4b (Huang et al., 2011; Leclercq et al., 2011; Lee et al., 2012).

Molecular serotyping showed that L. monocytogenes strains belonging to Group 1 (1/2a and 3a) were more commonly encountered in pork sausage (45.8%) and chicken leg (41.9%), whereas strains belonging to Group 3 (1/2b, 3b, and 7) were more frequent in hot dog (41.8%). In ground beef, the majority of the strains (33.7%) presented an atypical behavior and were classified as nontypeable. Considering that these strains are possibly of serotype 4b, which is the most common serotype associated with human listeriosis outbreaks (Huang et al., 2010; Leclercq et al., 2011; Lee et al., 2012), this result highlights the risk of consumption of undercooked ground meat.

The serotyping is an important epidemiological tool for the screening and characterization of L. monocytogenes. The wide diversity of L. monocytogenes serotypes in meat products found in this study has been reported by other authors in Brazil. Miyazaki et al. (2009) reported the prevalence of serotypes 4a and 4c (65.5%) in pork sausages. In hot dogs, Pettinati et al. (2006) reported that the most frequent serotypes were 1/2a (41.2%) and 1/2c (41.2%), while Monteiro et al. (2013) observed the prevalence of serotypes 4b and 1/2b in ground beef.

The analytical method used to search for L. monocytogenes played an important role in the detected prevalence in the tested samples. The methods of detection (ISO 11290-1:1996/Amd.1:2004) and the method of enumeration (ISO 11290-2:1998) did not perform equally in identifying the positive samples. In 25 samples of ground beef, 5 of chicken leg, 3 of pork sausage, and 2 of hot dog, which corresponds to 13% of the positive samples, L. monocytogenes was detected only when the enumeration method was used. According to Bruhn et al. (2005), the enrichment steps favor the growth of other Listeria species, such as L. innocua, increasing the difficulty of isolating L. monocytogenes. Moreover, some authors suggest that L. innocua can produce inhibitory components that hinder the detection of L. monocytogenes when the contamination is low (Cornu et al., 2002). The use of the two methods simultaneously and plating onto two selective media (ALOA and Palcam) could explain the higher prevalence than that reported in other Brazilian studies and certainly contributed to a better assessment of the presence of L. monocytogenes in the tested food products.

These data on prevalence, level of contamination, and most frequent serotypes of L. monocytogenes of meat products in Sao Paulo are useful for future risk assessments and relevant for establishing science-based intervention strategies aimed at reducing these risks, especially for pregnant women and immunocompromised individuals.

Footnotes

Acknowledgments

The present work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (Projects no. 07/54650-5). We thank COVISA (SP) for the samples.

Disclosure Statement

No competing financial interests exist.

References

Allerberger

, Wagner

. Listeriosis: A resurgent foodborne infection. Clin Microbiol Infect, 2010; 16:16–23.

Barros

, Nero

, Silva

, d'Ovidio

, Monteiro

, Tamanini

, Fagnani

, Hofer

, Beloti

. Listeria monocytogenes: Occurrence in beef and identification of the main contamination points in processing plants. Meat Sci, 2007; 76:591–596.

Blum-Menezes

, Deliberalli

, Bittencourt

, Couto

, Barbosa

, Santos

, Pinto

. Listeriosis in the far South of Brazil: Neglected infection?. Rev Soc Bras Med Trop, 2013; 46:381–383.

Bruhn

, Vogel

, Gram

. Bias in the Listeria monocytogenes enrichment procedure: Lineage 2 strains outcompete lineage 1 strains in University of Vermont selective enrichments. Appl Environ Microbiol, 2005; 71:961–967.

Cabedo

, Picart

, Barrot

, Teixido

, Canelles

. Prevalence of Listeria monocytogenes and Salmonella in ready-to-eat food in Catalonia, Spain. J Food Prot, 2008; 71:855–859.

Cartwright

, Jackson

, Johnson

, Graves

, Silk

, Mahon

. Listeriosis outbreaks and associated food vehicles, United States, 1998–2008. Emerg Infect Dis, 2013; 19:1–9.

[CDC] Centers for Disease Control and Prevention. Vital signs: Listeria illnesses, deaths, and outbreaks–United States, 2009–2011. MMWR Morb Mortal Wkly Rep, 2013; 62:448–452.

Chen

, Zhang

, Mei

, Jiang

, Fang

. Prevalence of Listeria in Chinese food products from 13 provinces between 2000 and 2007 and virulence characterization of Listeria monocytogenes isolates. Foodborne Pathog Dis, 2009; 6:7–14.

Cornu

, Kalmokoff

, Flandrois

. Modelling the competitive growth of Listeria monocytogenes & Listeria innocua in enrichment broths. Int J Food Microbiol, 2002; 73:261–274.

10.

Derra

, Karlsmose

, Monga

, Mache

, Svendsen

, Félix

, Granier

, Geyid

, Taye

, Hendriksen

. Occurrence of Listeria spp. in retail meat and dairy products in the area of Addis Ababa, Ethiopia. Foodborne Pathog Dis, 2013; 10:577–579.

11.

Doumith

, Buchrieser

, Glaser

, Jacquet

, Martin

. Differentiation of the major Listeria monocytogenes serovars by multiplex PCR. J Clin Microbiol, 2004; 42:3819–3822.

12.

Fox

, deLappe

, Garvey

, McKeown

, Cormican

, Leonard

, Jordan

. PFGE analysis of Listeria monocytogenes isolates of clinical, animal, food and environmental origin from Ireland. J Med Microbiol, 2012; 61:540–547.

13.

Hof

. Listeria monocytogenes: A causative agent of gastroenteritis?. Eur J Clin Microbiol Infect Dis, 2001; 20:369–373.

14.

Huang

, Fang

, Dimovski

, Wang

, Hogg

, Bates

. Observation of a new pattern in serogroup-related PCR typing in Listeria monocytogenes 4b isolates. J Clin Microbiol, 2011; 49:426–429.

15.

[IBGE] Instituto Brasileiro de Geografia e Estatística. Cidades. São Paulo, SP. Infográficos. 2009. Available at: http://www.ibge.gov.br/cidadesat/topwindow.htm, accessed December 12, 2009 . (In Portuguese.)

16.

[ISO] International Organization for Standardization. ISO 11290-1: Microbiology of Food and Animal Feeding Stuffs: Horizontal Method for the Detection and Enumeration of Listeria monocytogenes. Part 1: Detection Method: Modification of the Isolation Media and the Haemolysis Test, and Inclusion of Precision Data. Zurich, Switzerland: ISO, 2004.

17.

ISO. ISO 11290-2:1998: Microbiology of Food and Animal Feeding Stuffs: Horizontal Method for the Detection and Enumeration of Listeria monocytogenes. Part 2: Enumeration Method . Zurich, Switzerland: ISO, 1998.

18.

Jemmi

, Stephan

. Listeria monocytogenes: Food-borne pathogen and hygiene indicator. Rev Sci Tech, 2006; 25:571–580.

19.

Khen

, Lynch

, Carroll

, McDowell

, Duffy

. Occurrence, antibiotic resistance and molecular characterization of Listeria monocytogenes in the beef chain in the Republic of Ireland. Zoonoses Public Health, 2014; 61:1–7.

20.

Leclercq

, Chenal-Francisque

, Dieye

, Cantinelli

, Drali

, Brisse

, Lecuit

. Characterization of the novel Listeria monocytogenes PCR serogrouping profile IVb-v1. Int J Food Microbiol, 2011; 147:74–77.

21.

Lee

, Ward

, Graves

, Wolf

, Sperry

, Siletzky

, Kathariou

. Atypical Listeria monocytogenes serotype 4b strains harboring a lineage II-specific gene cassette. Appl Environ Microbiol, 2012; 78:660–667.

22.

Martins

, Germano

PML

. Listeria monocytogenes in ready-to-eat, sliced, cooked ham and salami products, marketed in the city of São Paulo, Brazil: Occurrence, quantification, and serotyping. Food Control, 2011; 22:297–302.

23.

Martins

, Faria

, Miguel

, Dias

, Cardoso

, Magalhães

, Mascarenhas

, Nouér

, Barbosa

, Vallim

, Hofer

, Rabello

, Riley

, Moreira

. A cluster of Listeria monocytogenes infections in hospitalized adults. Am J Infect Control, 2010; 38:e31–e36.

24.

Meloni

, Galluzzo

, Mureddu

, Piras

, Griffiths

, Mazzette

. Listeria monocytogenes in RTE foods marketed in Italy: Prevalence and automated EcoRI ribotyping of the isolates. Int J Food Microbiol, 2009; 129:166–173.

25.

Miyasaki

, Chiarini

, Santa Ana Ade

, Destro

, Landgraf

, Franco

. High prevalence, low counts and uncommon serotypes of Listeria monocytogenes in linguiça, a Brazilian fresh pork sausage. Meat Sci, 2009; 83:523–527.

26.

Modzelewska-Kapituła

, Maj-Sobotka

. The microbial safety of ready-to-eat raw and cooked sausages in Poland: Listeria monocytogenes and Salmonella spp. occurrence. Food Control, 2014; 36:212–216.

27.

de Monteiro

LRL

, de Mesquita

, André

MCDPB

, Cardoso

. Molecular characterization of Listeria monocytogenes isolated from animal products in a city of Northern Brazil. Ciênc Rural, 2013; 43:1443–1448.

28.

Nørrung

. Microbiological criteria for Listeria monocytogenes in foods under special consideration of risk assessment approaches. Int J Food Microbiol, 2000; 62:217–221.

29.

Pao

, Ettinger

. Comparison of the microbial quality of ground beef and ground beef patties from internet and local retail markets. J Food Prot, 2009; 72:1722–1726.

30.

Pettinati

, Telles

, Balian

. Listeria monocytogenes in hot dog sausage obtained from groceries stores in the city of São Paulo: A comparative and retrospective analysis of human listeriosis isolations. Vet Zootec, 2006; 13:182–191.

31.

Pontello

, Guaita

, Sala

, Cipolla

, Gattuso

, Sonnessa

, Gianfranceschi

. Listeria monocytogenes serotypes in human infections (Italy, 2000–2010). Ann Ist Super Sanita, 2012; 48:146–150.

32.

Pouillot

, Hoelzer

, Jackson

, Henao

, Silk

. Relative risk of listeriosis in Foodborne Diseases Active Surveillance Network (FoodNet) sites according to age, pregnancy, and ethnicity. Clin Infect Dis, 2012; 54:405–410.

33.

Praakle-Amin

, Hanninen

, Korkeala

. Prevalence and genetic characterization of Listeria monocytogenes in retail broiler meat in Estonia. J Food Prot, 2006; 69:436–440.

34.

Rocourt

, Jacquet

, Reilly

. Epidemiology of human listeriosis and seafoods. Int J Food Microbiol, 2000; 62:197–209.

35.

Siegel

. Estatística Não Paramétricas Para As Ciências do Comportamento. São Paulo: McGraw-Hill do Brasil, 1975. (In Portuguese.)

36.

Simpson Beauchamp

, Byelashov

, Geornaras

, Kendall

, Scanga

, Belk

, Smith

, Sofos

. Fate of Listeria monocytogenes during freezing, thawing and home storage of frankfurters. Food Microbiol, 2010; 27:144–149.

37.

Swaminathan

, Gerner-Smidt

. The epidemiology of human listeriosis. Microbes Infect, 2007; 9:1236–1243.

38.

Thevenot

, Dernburg

, Vernozy-Rozand

. An updated review of Listeria monocytogenes in the pork meat industry and its products. J Appl Microbiol, 2006; 101:7–17.

39.

Vazquez-Boland

, Dominguez-Bernal

, Gonzalez-Zorn

, Kreft

, Goebel

. Pathogenicity islands and virulence evolution in Listeria . Microbes Infect, 2001; 3:571–584.

40.

Vivant

A-L

, Garmyn

, Piveteau

. Listeria monocytogenes, a down-to-earth pathogen. Front Cell Infect Microbiol, 2013; 87:1–10.

41.

Zhang

, Wang

, Xia

, Yang

, Xi

, Meng

. Isolation and characterization of Listeria monocytogenes isolates from retail foods in Shaanxi Province, China. Foodborne Pathog Dis, 2013; 10:867–872.

42.

Zhu

, Feng

, Zhang

, Zhu

, Luo

. Prevalence and serotypes of Listeria monocytogenes contamination in Chinese beef processing plants. Foodborne Pathog Dis, 2012; 9:556–560.