Letters in Applied Microbiology 1992, 15, 168-171

Listevia monocytogenes serotypes in Italian meat products

G. COMI, R . FRIGERIO* & C. CANTONI* Dipartimento di Scienze4limentario, Facolta di Agraria, Universita di Udine, Via Marangoni 97,33100 Udine and *Istituto di lspezione degli Alimenti de Origine Animale, Facolta d i Medicina Veterinaria, Universita d i Milano, Italy

PGBI08: received 16 June 1992 and accepted 18 June I992

C O M I , G . , F R I G E R I O , R . & C A N T O N I , C. 1992. Listeria monocytogenes serotypes in Italian meat products. Letters in Applied Microbiology 15, 168-171.

Listeria monocytogenes was isolated and enumerated in Italian fresh ground beef, fresh pork meat and industrial sausages. All the samples contained less than 2000 L. monocytoyenes/g of meat. The main serotype isolated was 1/2c (56.9%). Other serotypes isolated included 1/2a, 1/2b, 3c, 4b and 4c. A prevalence of less virulent serotypes over more virulent was thus noted. It seems that the low incidence of listeriosis from these products is related to the low concentration and virulence of L. rnonocytogenes present.

Listeria spp. are widespread in nature and have been isolated from soil, vegetable and animal tissues, insilates, water and foods (Gray 1966; Welshimer & Voet 1971; Weis & Seeliger 1975; Schlech et al. 1983; Billaux 1990; Dickson 1990).

Of the different species of listeria only L. monocytogenes has become recognized as an important foodborne pathogen, humans and animals being highly sensitive to L. mono- cytogenes.

The main cases are described in Switzerland between 1981 and 1987 and in North America (Fleming et al. 1985; Linnan et al. 1987; Anon. 1988; Bille & Glauser 1988). In the USA spor- adic cases have been described every year, from 1983 until today, involving 300 people with 100 fatalities (Farber & Peterkin 1991). Generally listeria have low pathogenicity and foodstuffs are considered a potential danger when they contain between 1ooO-1OOOOO00 cfu/g of L. monocytogenes (Farber & Peterkin 1991).

Low numbers of this micro-organism can produce the disease only in persons in high risk categories (diabetics, infants, old people, cancer and AIDS patients).

In the cases reported in California, the affected people belonged to a category defined

as ‘at high risk of infection’ and were mainly represented by pregnant women who transmit- ted listeriosis to their unborn children, of whom 21 were born dead or died after birth.

Listeria monocytogenes has been isolated in many foods, including raw meats, milk and soft cheese (Nicolas et al. 1989; Comi et al. 1990a, b) and is able to survive for weeks or months, in dry or humid places and in cultivated soil.

This micro-organism can also develop at low temperature and is tolerant of some preservative agents such as NaCl and NaOH, , which makes it particularly dangerous as a polluting agent for refrigerated foods after their production (Cantoni et al. 1988). Although only sporadic cases have been reported from meat and its derivatives, the potential risk from the con- sumption of processed meat is still being studied (Ryser & Marth 1991; Farber & Peterkin 1991).

Listeria monocytogenes has been isolated from pork meat, ground beef, sausages, minced meat and delicatessen products (Lee & McClain 1986; Le Guillox 1980; Nicolas et al. 1989; Cantoni et al. 1989; Barbuti et al. 1990; Billaux 1990; Dickson 1990). It was not isolated from healthy cow muscles, but was found in the lungs (50% of cases) and in the spleen (87% of cases) (Cottin et al. 1985).

Listeria monocytogenes in meat products 169 Table 1. Number of samples with or without Listeria monocytogenes

Listeria Other Negative Type of meat No. of samples rnonocytoqenes listeria samples

Fresh pork 153 24 29 100

Sausages 189 27 83 79 Fresh ground beef 148 14 64 70

Total samdes 490 65 176 249

Spread of the bacterium in meat products was increased by the milling of meat and its manipu- lation (Le Guillox 1980; Cantoni et al. 1989). However, listeria does not seem to increase in number during ripening of meat or sausage (Shahamat et nl. 1980; Johnson et al. 1988; Gafner et al. 1989). In fact the pH value, addi- tives, organic acids and the presence of other bacteria can inhibit listeria growth (Comi et al. 1990c) and consequently L. monocytogenes never seems to reach such high concentrations as to become a pathogenic hazard to con- sumers.

In order to assess whether a product is of a high hygienic level both the presence and con- centration of the serotypes must be known. Serotypes 1/2a, 1/2b abd 4b are known to be particularly virulent and 90% of listeriosis out- breaks are caused by these serotypes. Even though other serotypes are present in food, they are much less virulent.

The aim of our work was to study the dis- tribution of L. monocytogenes serotypes in selec- ted Italian meats.

Materials and Methods

Four hundred and ninety samples of pork, ground beef meat and sausages were examined using Listeria Enrichment broth (Biolife, Milan) and Palcam agar (van Netten et al. 1989) to determine presence or absence of listeria.

The direct plate count technique was used to enumerate listeria in positive samples by the enrichment technique. To 10 g of sample we added 90 ml of sterile peptone water (1 : 10) and homogenized the mixture with a Stomacher (PBI; Italy). One ml of decimal dilution was dis- tributed in five Palcam agar plates, taking care to 'plate' the inoculum on all the surface of the medium. The plates were incubated for 48 h at 30°C under reduced oxygen concentration.

Suspect listeria colonies were identified by : Gram strain and motility; fermentation of L-

rhamnose, D-xylose and mannitol ; catalase, hae- molysis, CAMP and urease tests. Confirmation was by the MICRO-ID system (Mastransia, Milan).

Serotypes were identified by Professor J. Bille and Dr E.S. Bannermann of National Research Centre for Listeria, at Institute of Microbiology in Lausanne (Centre Hospitalier Universitaire Vadois), Switzerland.

Results

Out of 490 samples of meat products, 241 (49.1%) were contaminated by Listeria spp., 65 (1 3.2%) of the samples being contaminated by L. monocytogenes, 176 (35.9%) by other listeria such as L. innocua, L. grayi, L. murrayi, L. welshimeri and L. iuanouii (Table 1).

Twenty-four samples of fresh ground beef, 14 of fresh pork meat and 27 of sausages contained L. monocytogenes. The concentration of L. monocytogenes in positive samples was very low (Table 2), all samples containing less than 2000 L. monocytogeneslg of the product.

From these results we can note that 91.6% of ground beef samples contained less than 100 L. monocytogeneslg; 5.6% less or equal to lOOO/g and 2.8% less or equal to 2OOO/g. At the same time 92.6% of pork meat contained less than 100 L. monocytogeneslg, 3.7% less or equal to 100O/g, and 3.7% less or equal to 2OOOlg.

The meat samples were analysed after 3-7 d of storage at 4"C, and at the end of their com- mercial life; sausages were analysed after 6-10 d

Table 2. Listeria monocyrogenes concentration in meat uroducts

Range Ground beef Pork meat < 100 91.6 9 2 6 100-1000 5.6 3.7 1000-2000 2.8 3.7

Total samples 100.0 100.0

Data in %, of positive samples.

G. Comi et al. Table 3. Listeria monocytogenes sero-

types from meat products

Serotypes Number %

1/2a 10 15.3 1/2b 7 10.7 1/2c 37 56.9 3c 2 3.1 4b 8 12.4 4c 1 1.6

Total strains 65 100.0

of their production. In none of the above cases did we observe an increase in the numbers of Listeria spp. The absence of growth during storage is due to the chemical-physical and microbiological characteristics of the products (water activity, NaCI, pH, fatty acids) and the antagonistic microflora (Johnson et al. 1988; Comi et a!. 1990~).

Table 3 shows the percentage of serotypes of L. monocytogenes isolated from meat product. Serotype 1/2c was more widespread than the others, 56.9% of L. monocytogenes belonging to this serotype. Serotype 1/2a (1 5.3%), serotype 4b (12.4%), serotype 1/2b (10.7%), serotype 3c (3.1%) and 4c (1.6%) were the remaining serotypes isolated.

Discussion

The incidence of L. monocytogenes in all meat samples analysed was low (13.2% of total samples). However the presence of Listeria spp. in meat products is very high, considering 49.1% positive samples were obtained. These results demonstrate once again that listeria is widespread in nature and can easily contam- inate meat products and other foods.

The presence of the L. monocytogenes in sea- soned sausages confirmed survival through several seasoning stages up to the moment of sale. This is in accordance with recent studies carried ou t by various authors (Gafner et al. 1989; Johnson et al. 1988) on the behaviour of L. monocytogenes during salami seasoning.

The results of the quantitative survey carried out using the direct plate count method, showed that only a low percentage of samples contained L. monocytogenes values over 100 cfu/g, below which level van Netten et al. (1989) suggested foods do not constitute a public health risk.

To determine values less or equal to 10 cfu/g, it is necessary to use the most probable number technique (MPN) (Comi et al. 1990b). Listeria spp. cannot be counted by direct count plate techniques in samples with high levels of con- taminating flora, since selective agars are not sufficiently selective, where a high number of yeasts, moulds, cocci and rods are present. In this case the selective plates are covered by a slime of micro-organisms and it is impossible to count Listeria. This is especially the case with samples of moulded sausages or cheeses. Sero- types, which are responsible for foodborne liste- riosis, were recovered in a low percentage.

Results obtained on the incidence of sero- types contained in meat were in accordance with McLauchlin (1990). There was a prevalence of the serotypes considered to be less virulent (1/2c, 3c, 4c; -59.2%) compared to those of a medium or high virulence (1/2a, 1/2b, 4b;

The absence of outbreaks of Iisteriosis from the products sampled in this study is probably due to the low concentration of L. mono- cytogenes present, the presence of serotypes with low pathogenicity and destruction of the organism on cooking fresh pork and fresh ground beef.

We thank Professor J. Bille and Dr E.S. Ban- nermann for serotyping the Listeria mono- cytogenes that we isolated.

-40.8%).

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