Listeria monocytogenes occurrence and characterizationin meat-producing plants

A. Peccio1, T. Autio2, H. Korkeala2, R. Rosmini1 and M. Trevisani11Dipartimento di Sanita Pubblica Veterinaria e Patologia Animale, Facolta di Medicina Veterinaria, Alma Mater Studiorum, Universita di

Bologna, Italy, and 2Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, University of Helsinki, Helsinki,

Finland

2003/0099: received 6 February 2003, revised 13 May 2003 and accepted 1 June 2003

ABSTRACT

A. PECCIO, T . AUTIO, H . KORKEALA, R . ROSMINI AND M. TREVISANI . 2003.

Aims: The prevalence, level of contamination and epidemiological profile of Listeria monocytogenes were

investigated in two meat-producing plants during a 20-month period.

Methods and Results: Sampling for L. monocytogenes was carried out in a cattle slaughterhouse (n ¼ 72) and

a swine meat-processing plant (n ¼ 68) during a 20-month period. Swabs and food samples were analysed with

the most probable number (MPN) technique for L. monocytogenes and the isolated strains were characterized by

AscI-restriction analysis pulsed-field gel electrophoresis (REA–PFGE). Contamination of meat and meat products

was always at low level (below 50 MPN per gram). The seven L. monocytogenes positive samples isolated in the

bovine slaughterhouse yielded strains with the same REA–PFGE profile. However, the seven strains isolated in

the swine meat processing plant showed six different profiles. Two of them showed indistinguishable profiles

with L. monocytogenes strains collected from other meat processing facilities located in the same area.

Significance and Impact of the Study: The genotyping method is a valuable tool to investigate contamination

sources. The study of REA–PFGE profiles indicated that environmental contamination was probably responsible

for the persistence of over 16 months of one strain of L. monocytogenes in the cattle slaughterhouse. Several

meat suppliers could be responsible for the contamination in the pig meat processing facility, and this is confirmed

by the finding of some identical strain in other meat processing facilities located in the same area.

Keywords: Listeria monocytogenes, beef slaughterhouse, epidemiological characterization, pork processing, REA –

PFGE.

INTRODUCTION

Occurrence of Listeria monocytogenes within slaughterhouses

and meat processing facilities has been associated with

environmental colonization, because of its ability to adapt

and survive even on ‘clean’ equipment and rooms (Lunden

et al. 2000). However, L. monocytogenes can enter through

infected animals and raw meat or intermediate products

processed by suppliers (Boerlin and Piffaretti 1991; Gill and

Jones 1995; Fenlon et al. 1996; Nesbakken et al. 1996;

Sammarco et al. 1997). This poses a microbiological risk in

products, which requires enforcement of specific control

measures. Strategies and control measures could be

improved if data on the prevalence and location (hot spot)

of Listeria within processing facilities is analysed. Charac-

terization of isolated strains is an essential tool in tracing

contamination sources and genotyping of L. monocytogenes

can be achieved by using pulsed-field gel electrophoresis

(PFGE) (Destro et al. 1996; Autio et al. 1999; Giovannacci

et al. 1999; Miettinen et al. 1999; Autio et al. 2000;

Chasseignaux et al. 2001). The aim of this study was to

determine the prevalence and the epidemiological profile of

Correspondence to: M. Trevisani, Dipartimento di Sanit�aa Pubblica Veterinaria e

Patologia Animale, Universit�aa di Bologna, via Tolara di Sopra 50, 40064 Ozzano

Emilia (BO), Italy (e-mail: trevisani@vet.unibo.it).

ª 2003 The Society for Applied Microbiology

Letters in Applied Microbiology 2003, 37, 234–238 doi:10.1046/j.1472-765X.2003.01384.x

L. monocytogenes in two EC-approved meat-producing plants

to find the correlation between environmental contamination

and occurrence of this food-borne pathogen in the final

products.

MATERIAL AND METHODS

Sampling

The samples were collected from two meat processing

facilities, a cattle slaughterhouse (plant A) and a pork

processing plant (plant B) from September 2000 to April

2002 (Tables 1 and 2). Both plants were located in the same

producing area in north-east Italy. Environment (fridge

rooms and drains), equipment (tables, saws, kneaders and

mincers) and utensils (knives) were sampled by swabbing a

20 cm2 area (46 from plant A and 51 from plant B). The

swabs were taken both during processing and/or before

starting the process, after the cleaning operations. Swabs

(n ¼ 14) were also taken to detect Listeria on cattle carcass

(i.e. from shoulder and abdominal incision), swine large

meat cuts (n ¼ 6) and from batches of minced meat (n ¼ 4).

Swabbing was performed using several sterile cotton swabs

moistened with NaCl 0Æ85%. After sampling, the swabs

were soaked in 2 ml of saline solution. Meat samples (12

from plant A and seven from plant B) were taken at different

phases of the processing. They included raw meat cuts,

minced meat prior to salting and unseasoned and uncooked

sausages that had to be cooked before eating. Meat samples

and swabs were stored at 0–8�C and were examined within

72 h of arrival.

Isolation and identification of L. monocytogenes

Samples were examined using the most probable number

(MPN) technique according to the Italian official method

(O.M. Sanit�aa 1993). Ten grams of each food sample was

homogenized in a stomacher with 90 ml of buffered peptone

water (Oxoid, Basingstoke, UK) for 4 min and two 10-fold

serial dilutions in 0Æ1% tryptone (Oxoid) were made. The

saline solution (2 ml) including the swabs was also used to

prepare 10-fold serial dilutions. One millilitre of each dilution

was aseptically added to a series of three tubes containing 9 ml

of Fraser enrichment broth (Oxoid). Tubes were incubated at

32�C for 48 h. The volumes (10 ll each) of the broth were

plated on Oxford agar (Oxoid) and incubated at 37�C for

48 h. Five suspected colonies from Oxford agar were picked

and streaked out on Tryptone soya agar (Oxoid) plus 0Æ6%

yeast extract (YE; Difco Becton Dickinson, Sparks, MD,

USA) and incubated at 37�C for 24 h. Suspected colonies

were streaked out on blood agar [blood agar base (Difco), 5%

sheep blood] and incubated at 37�C for 24 h. b-haemolytic

colonies were further identified as L. monocytogenes by testing

motility at 25�C, catalase reaction, carbohydrates acid reac-

tion of rhamnose 0Æ5% (Sigma), xylose 0Æ5% (Sigma) and

mannitol 1% (Sigma) in purple broth base (Difco). Two

isolates for each positive sample were stored at )20�C in

Tryptone soya broth (Oxoid) + 20% glycerol (Sigma).

DNA analysis by PFGE

Cultures for DNA isolation were grown overnight in brain

heart infusion (BHI) broth (Oxoid) at 37�C. DNA was

Table 1 Level, prevalence and characteriza-

tion of Listeria monocytogenes contamination in

raw meat, carcasses, environmental and

equipment samples in a bovine slaughterhouse

plant ASource

No. of L. monocytogenes

positive/no. of samplesContamination level

(MPN) AscI REDPDuring process After cleaning

Meat

Excised samples 3/12 3Æ6; 3Æ6 and 43 g)1 1, 1, 1

Carcass swabs 1/14 0.33 cm)2 1

Environment and equipment

Knives 2/6 1/5 0Æ33* 9Æ2* and 0Æ46� cm)2 1, 1, 1

Fridge room 0/4 0/4

Tables 0/4 0/5

Saws 0/4 0/4

Floor drains 0/5

Others 0/2 0/3

Total 6/51 1/21

*During process.

�Cleaned sites.

MPN: most probable number; REDP, restriction enzyme digestion profiles.

LISTERIA IN MEAT PLANTS 235

ª 2003 The Society for Applied Microbiology, Letters in Applied Microbiology, 37, 234–238, doi:10.1046/j.1472-765X.2003.01384.x

isolated and digested with AscI restriction enzyme (New

England Biolabs, Beverly, MA, USA) in agarose plugs and

characterized by PFGE as described by Autio et al. (1999).

Isolates which displayed indistinguishable restriction

enzyme digestion profiles (REDP) were placed in the same

group, whereas isolates that differed by one or more bands

were considered different (Miettinen et al. 1999). Patterns

were also compared with those of other six strains isolated

from different meat processing facilities located in the same

area (Fig. 1).

RESULTS

Listeria monocytogenes was detected in four of the 26 cattle

carcasses sampled in plant A with contamination level

between 3Æ6 and 43 MPN per gram in meat samples and

0Æ33 cm)2 on carcass swabs. Presence of L. monocytogeneswas also detected in three of the 11 knife swabs, including

one that was presumed to be ‘clean’, but was not in the

fridge room, drain, tables and saws. The level of contam-

ination on knives was between 0Æ33 and 9Æ2 MPN per cm2

(Table 1).

In the pork processing facility (plant B), L. monocytogenes

was isolated from uncooked end products, partially proc-

essed (salted minced meat mixes with nitrites and spices),

and swine raw meat from suppliers. Finished products like

raw sausages (country style and cotechino) were more

frequently contaminated. Positive samples showed contam-

ination level between 3Æ6 and 23 MPN per gram. Contam-

ination by L. monocytogenes was found on kneader and on

mincer, but not on stuffers, tables and other surfaces in

contact with meat. It was also observed that the contamin-

ated mincer was presumed to be ‘clean’. Contamination level

was 0Æ184 MPN cm)2 in both the positive swabs (Table 2).

The PFGE analysis showed 11 AscI pulsotypes charac-

terized by 9–12 fragments between 23 and 486Æ5 kb (Fig. 1).

All the seven L. monocytogenes strains collected in plant A

showed the same pulsotype (REDP-1), while the seven

strains collected in plant B had six different profiles. Two

strains with the same profile (REDP-4) were isolated from

sausages at different times. Two pulsotypes (REDP-2 and

REDP-7) were also found among the strains isolated from

other meat processing facilities located in the same area.

DISCUSSION

Listeria monocytogenes was isolated with relatively high

frequency in meat products, but concentration was low,

below the unacceptable limits set in Italy (110 cells per

gram) and other European countries (100 cells per gram) for

products intended to be consumed after cooking. However,

the occurrence of this pathogen in food processing facilities

must be investigated. Many authors have proved the

widespread occurrence of this pathogen in raw meat (e.g.

McLauchlin 1996, Nørrung et al. 1999). Some authors

emphasized on environmental conditions for the spread of

L. monocytogenes contamination (Nesbakken et al. 1996),

others have focused on the equipments used as points of

L. monocytogenes cross-contamination (Gill and Jones 1995;

Autio et al. 2000). In both plants, L. monocytogenes was

detected in equipment and tools occasionally at a very low

level. It was isolated from kneader and mincers, used in the

Table 2 Level, prevalence and characteriza-

tion of Listeria monocytogenes contamination in

meat products, environmental and equipment

samples in a pork processing plant BSource

No. of L. monocytogenes

positive/no. of samplesContamination

level (MPN) AscI REDPDuring process After cleaning

Meat products

Swine raw meat 1/2 3Æ6 g)1 5

Swine raw meat swabs 0/6

Minced meat for sausage 1/1 9Æ2 g)1 2

Swine minced meat swabs 0/4

Cotechino 2/2 3Æ6 and 21 g)1 4, 6

Country-style sausage 1/2 23 g)1 4

Environment and equipment in the sausage processing room

Kneader 1/3 0/6 0Æ184 cm)2 7

Mincer 0/2 1/6 0Æ184 cm)2 11

Stuffer 0/3 0/8

Tables 0/3 0/6

Others 0/7 0/7

Total 6/35 1/33

MPN: most probable number; REDP, restriction enzyme digestion profiles.

236 A. PECCIO ET AL.

ª 2003 The Society for Applied Microbiology, Letters in Applied Microbiology, 37, 234–238, doi:10.1046/j.1472-765X.2003.01384.x

pig meat processing facility, and on knives, used in the beef

slaughterhouse. However, it was never found in fridge

rooms and in floor drains. It was found that L. monocytogenes

is able to persist after the cleaning procedures. Indeed, it was

detected on a mincer, supposed to be clean, and on a knife

cleaned and left in the sterilizing tank. In plant A, all seven

strains isolated from different samples (three excised meat

samples, one carcass swab and three knife swabs) showed the

same AscI restriction profile (REDP-1). The isolates were

detected from samples taken at five different times, over

16 months. Twice cross-contamination through the knives

used for de-hiding operations was pointed out, because in

two cases isolates were collected at the same time from knife

and carcass swabs or from the forelegs. Although a common

source cannot be identified, these results showed that a

unique strain could persist. This result is consistent with

studies of other researchers who indicated that slaughtering

equipment and environment are probable sites of cross-

contamination even for several months (Giovannacci et al.

1999; Miettinen et al. 1999; Autio et al. 2002).

Differently, seven strains with six different AscI restric-

tion profiles were isolated in the pork processing plant. Only

strain REDP-4 was detected twice. The results suggest that

raw materials, which are supplied by several producers, may

play a major role as a source of contamination of the plant. It

cannot be neglected that the cleaning operations were not

effective in eliminating L. monocytogenes, as shown by an

isolate recovered from a cleaned mincer. Among the isolates

collected at this plant, two showed a restriction enzyme

digestion profile (REDP-2 and REDP-7) identical to other

strains of our collection recovered at that time in different

plants and retail shop from the same region. This finding

may suggest these genotypes could be widespread within a

geographical region or food industry environment and

should be further investigated.

REFERENCES

Autio, T., Hielm, S., Miettinen, M., Sjoberg, A.-M., Aarnisalo, K.,

Bjorkroth, J., Mattila-Sandholm, T. and Korkeala, H. (1999)

Sources of Listeria monocytogenes contamination in a cold-smoked

rainbow trout processing plant detected by pulsed-field gel

electrophoresis typing. Applied and Environmental Microbiology 65,

150–155.

Autio, T., Sateri, T., Fredriksson-Ahomaa, M., Rahkio, M., Lunden,

J. and Korkeala, H. (2000) Listeria monocytogenes contamination

pattern in pig slaughterhouses. Journal of Food Protection 63, 1438–

1442.

Autio, T., Lunden, J., Fredriksson-Ahomaa, M., Bjorkroth, J.,

Sjoberg, A.-M. and Korkeala, H. (2002) Similar Listeria monocyto-

genes pulsotypes detected in several foods originating from different

sources. International Journal of Food Microbiology 77, 83–90.

Boerlin, P. and Piffaretti, J.C. (1991) Typing of human, animal, food,

and environmental isolates of Listeria monocytogenes by multilocus

enzyme electrophoresis. Applied and Environmental Microbiology 57,

1624–1629.

Chasseignaux, E., Toquin, M.-T., Ragimbeau, C., Salvat, G., Colin, P.

and Ermel, G. (2001) Molecular epidemiology of Listeria monocy-

togenes isolates collected from the environment, raw meat and raw

products in two poultry- and pork-processing plants. Journal of

Applied Microbiology 91, 888–899.

Destro, M.T., Leitao, M.F.F. and Farber, J.M. (1996) Use of

molecular typing methods to trace the dissemination of Listeria

monocytogenes in a shrimp processing plant. Applied and Environ-

mental Microbiology 62, 705–711.

Fenlon, D.R., Wilson, J. and Donachie, W. (1996) The incidence and

level of Listeria monocytogenes contamination of food sources at

primary production and initial processing. Journal of Applied

Bacteriology 81, 641–650.

Gill, C.O. and Jones, T. (1995) The presence of Aeromonas, Listeria

and Yersinia in carcass processing equipment at two pig slaughtering

plants. Food Microbiology 12, 135–141.

1 M 2 3 4 5 M 6 7 8 9 M 10

kb

436·5

388·0

291·0

242·5

194·0

145·0

97·0

48·5

23·0

Fig. 1 AscI restriction enzyme digestion profile (REDP) of Listeria

monocytogenes strains. Lanes: M, low range PFGE marker; 1, REDP-4;

2, REDP-9*; 3, REDP-10*; 4, 6, 8 and 9, REDP-1; 5, REDP-4; 7,

REDP-11; 10, REDP-8*. *REDPs of L. monocytogenes strains belong to

our collection

LISTERIA IN MEAT PLANTS 237

ª 2003 The Society for Applied Microbiology, Letters in Applied Microbiology, 37, 234–238, doi:10.1046/j.1472-765X.2003.01384.x

Giovannacci, I., Ragimbeau, C., Queguiner, S., Salvat, G., Vendeuvre,

J.-L., Carlier, V. and Ermel, G. (1999) Listeria monocytogenes in pork

slaughtering and cutting plants use of RAPD, PFGE and PCR-REA

for tracing and molecular epidemiology. International Journal of Food

Microbiology 53, 127–140.

Lunden, J.M., Miettinen, M.K., Autio, T.J. and Korkeala, H.J. (2000)

Persistent Listeria monocytogenes strains show enhanced adherence to

food contact surface after short contact times. Journal of Food

Protection 63, 1204–1207.

McLauchlin, J. (1996) The role of the public health laboratory Service

in England and Wales in the investigation of human listeriosis during

the 1980s and 1990s. Food Control 7, 235–239.

Miettinen, M.K., Bjorkroth, K.J. and Korkeala, H. (1999) Character-

ization of Listeria monocytogenes from an ice cream plant by

serotyping and pulsed-field gel electrophoresis. International Journal

of Food Microbiology 46, 187–192.

Nesbakken, T., Kapperud, G. and Caugant, D.A. (1996) Pathways of

Listeria monocytogenes contamination in the meat processing indus-

try. International Journal of Food Microbiology 31, 161–171.

Nørrung, B., Andersen, J.K. and Schlundt, J. (1999) Incidence and

control of Listeria monocytogenes in foods in Denmark. International

Journal of Food Microbiology 53, 195–203.

O.M. Sanit�aa (1993) Limiti di Listeria monocytogenes in alcuni prodotti

alimentari. Gazzetta Ufficiale della Repubblica Italiana – Serie

Generale 291, 18–20.

Sammarco, M.L., Ripabelli, G., Ruberto, A., Iannitto, G., and Grasso,

G.M. (1997) Prevalence of salmonellae, listeriae and yersiniae in the

slaughterhouse environment and on work surfaces, equipment, and

workers. Journal of Food Protection 60, 367–371.

238 A. PECCIO ET AL.

ª 2003 The Society for Applied Microbiology, Letters in Applied Microbiology, 37, 234–238, doi:10.1046/j.1472-765X.2003.01384.x