listeria monocytogenes occurrence and characterization in meat-producing plants
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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.
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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
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