Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

The evaluation of the microbial safety of fresh ready-to-eat

vegetables produced by different technologies in Italy
M. De Giusti1, C. Aurigemma1, L. Marinelli1, D. Tufi1, D. De Medici2, S. Di Pasquale2, C. De Vito1
and A. Boccia1
1 Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy
2 Department of SPVSA, Unit of Microbiological Foodborne Hazards, National Institute of Health, Rome, Italy

Keywords
bacterial contamination, GAP-GMP-HACCP
integrated system, microbial food safety,
ready-to-eat vegetables, viral contamination.
Correspondence
Maria De Giusti, Department of Experimental
Medicine, Sapienza University of Rome, Viale
Regina Elena 324, 00161 Rome, Italy.
E-mail: maria.degiusti@uniroma1.it

2010 2131: received 11 December 2009,

revised 8 March 2010 and accepted 12
March 2010
doi:10.1111/j.1365-2672.2010.04727.x

Abstract
Aims: The study was performed to evaluate the safety of whole and RTE vegetables and to investigate the effectiveness of different preventive strategies for
the quality assurance of RTE vegetables collected from three Italian production
systems. Producer 1, applied a strict system in compliance with GAP- GMP
HACCP, Producer 2 used chlorine disinfection at a second washing step, and
Producer 3 using a physical microbial stabilization.
Methods: During the period 20052007, a total of 964 samples including whole
vegetables and RTE salads, collected from three different producers in central
Italy, were analysed to quantify the aerobic mesophilic count (AMC) and
Escherichia coli, and for the presence of Salmonella spp, Listeria monocytogenes,
E. coli O157:H7, hepatitis A virus and Norovirus (NoV).
Results: None of the whole vegetable samples were positive for L. monocytogenes, E. coli O157:H7, HAV and NoV; however, a low prevalence of Salmonella was found. No pathogens were detected with cultural methods in any of
the RTE vegetables analysed, only two RTE samples were positive for L. monocytogenes with PCR, but were not confirmed by the cultural method. The
median values of AMC in RTE vegetables measured 24 h after packaging were
statistically different among the 3 producers (54 106, 15 107 and
37 107 CFU g)1, respectively; P = 0011). The lowest level was detected in
Producer 1.
Conclusion: The products that were processed applying rigorously GAP, GMP
and HACCP showed a better microbiological quality than those processed with
chemical or physical stabilization.
Study Significance and Impact: The results of the study evidenced the efficacy
of GAP, GMP and HACCP in improving microbiological quality of whole and
RTE vegetables.

Introduction
The role of fresh fruits and vegetables in a healthy diet is
well recognized in nutrition research as it ensures an adequate intake of vitamins, minerals, fibres and antioxidants
(Giugliano and Esposito 2008). The increasing demand
for fresh vegetables and for convenience food has resulted
in a growth of the market share for fresh ready-to-eat
(RTE) vegetables.
996

In Italy, the consumption of RTE vegetables has shown

a significant increase from 14 736 tonnes (138 677
million) in 2002 to 50 723 tonnes (397 599 million) in
2009 (ISMEA 2009). These products received some degree
of minimal technological processing before commercial
distribution. This processing, in most cases, is inadequate
in ensuring sterility or even microbiological stability. The
initial quality and subsequent handling of these products
appear to strongly influence the microbiology safety and

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M. De Giusti et al.

shelf life (Legnani and Leoni 2004). The factors that may
affect the occurrence of pathogen contaminations in vegetables include poor agricultural water quality, the use of
manure as a fertilizer and the incorrect application of
Good Manufacturing Practice (GMP) and Hazard Analysis and Critical Control Point (HACCP) systems during
production, processing, packaging and distribution.
The presence of pathogenic micro-organisms in these
products, mainly in leafy green vegetables, constitutes a
potential problem for consumer health and is a public
health issue (FAO WHO, 2008). The presence and prevalence of pathogenic micro-organisms in RTE salads have
been recognized in previous studies (Beuchat 1996; Wells
and Butterfield 1997; de Curtis et al. 2002; Froder et al.
2007; Little et al. 2007). Numerous epidemic outbreaks
because of the consumption of raw vegetables contaminated by pathogenic micro-organisms, such as Salmonella
spp, Escherichia coli O157, Listeria monoctogenes, hepatitis
A (HAV) and Norovirus (NoV) have been reported
(WHO, 2008).
In 2008, the European Rapid Alert System (RASFF)
received 99 alerts, 80 of which were reported from other
EU member states, while the remaining alerts were
received by the Italian National Alert System. Most alerts
were for fruit and vegetables (16%), and microbiological
hazards related to the presence of Salmonella (13 alerts),
Listeria (11 alerts) and E. coli (8 alerts) (RASFF, 2008). In
different years (20042006 and 2008), Salmonella contamination of Italian rucola lettuce was reported by the RASFF (Nygard et al. 2008).
Recently, microbiological criteria to evaluate food
hygiene conditions and HACCP performance of producers have been published (EC Regulation no.1441 2007
amending EC Regulation no. 2073 2005). This Regulation stipulates the recovery of Escherichia coli in RTE
vegetables, as an index of the hygienic process, and Salmonella spp and Listeria monocytogenes, as an index of
safety.
Although there are no mandatory microbiological criteria, which include reference limits for aerobic mesophilic
count (AMC), this parameter could represent a useful
tool to evaluate the microbiological quality of the production processes (Gelosa 1998; MD 22 mars 1993; PHLS
Guidelines 2000).
The aims of this study were to evaluate the microbial
hazards in whole and RTE vegetables and also to investigate the effectiveness of different production systems to
assure the safety and microbiological quality of vegetables. The entire food chain (from field to commercial
distribution) of these products was investigated using
different microbiological parameters [AMC, E. coli, Salmonella spp, L. monocytogenes, E. coli O157:H7, HAV
and NoV].

Italian RTE salads safety

Materials and methods

Samples
A total of 964 samples of whole vegetables and RTE
salads were analysed for AMC, E. coli, Salmonella spp,
L. monocytogenes and E. coli O157:H7, during the period
20052007. The samples analysed included 265 (275%)
whole vegetables (68 samples of endive, 56 of curly endive, 62 of rucola lettuce, 50 of red chicory, 23 of valerian
and 6 of red lettuce) and 699 (725%) RTE vegetables
[138 samples of endive, 102 of curly endive, 186 of rucola
lettuce, 12 of red chicory, 69 of valerian, 174 of mixed
salads (endive, rucola lettuce and red chicory), 18 of
mixed lettuce (white and red lettuce)]. One hundred
twenty-four of the 964 samples were also analysed for
HAV and NoV. The samples analysed included 46 whole
vegetables (16 samples of endive, 10 of curly endive, 10 of
rucola lettuce and 10 of red chicory) and 78 RTE salads
[30 samples of mixed salads (endive, rucola lettuce and
red chicory), 24 of endive, 24 of rucola lettuce].
Whole and RTE vegetable samples, stored at 4C and
packaged in polyethylene antifog bags (250 g), without a
modified atmosphere were randomly collected from three
different producers located in central Italy (one in
Campania and two in the Lazio region).
Production chains
The main steps of the production processes of the three
different producers include the receiving and storage of
the whole vegetable, the macroscopic inspection, the prewashing and preparation (cutting where appropriate), two
washing steps and the drying and packaging of the RTE
vegetables (Varzakas and Arvanitoyannis 2008). A brief
description of the three different production processes:
1. Producer 1: the food chain checks include an integrated system with strict compliance with GAP (Good
Agricultural Practice), GMP and HACCP and no microbial stabilization. The process included the washing of
whole vegetables after harvesting, cutting their lower
extremity and discarding all excess external leaves;
2. Producer 2: the food chain checks include chemical
microbial stabilization (chlorine disinfection at the second
washing step, 2% chlorine without an additional washing
step);
3. Producer 3: the food chain checks include physical
microbial stabilization using a tunnel freezer that permits
the rapid reduction in product temperature to +4C
applied after centrifugation and before packaging.
Producer 1 has a larger processing output, considering
the distribution area (Italy and other EU countries) than
Producer 2 and 3 (distribution only in Italy).

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M. De Giusti et al.

Microbiological analysis
All samples were transported at refrigeration temperature
(46C) to the laboratory. Whole vegetables and RTE
vegetable samples were immediately analysed (T0: at the
second day after harvesting; T1: at the first day after
packaging). RTE samples were stored at 4C and analysed
at the fifth day (Producer 1) and at the seventh day
(Producer 2 and 3) after packaging corresponding to the
expiration date (T2) and 2 days after the expiration date
(T3).
The microbiological determination using cultural and
molecular methods was performed using the standard
methodologies described in Table 1. All microbiological
media were from Oxoid (Cambridge, UK), unless otherwise specified.
Bacterial determination
Standard cultural method
Ten grams of each sample was diluted in 90 ml of buffered peptone water (BPW) and homogenized for 2 min
at 260 rev min)1 using a Stomacher (Model 400 circulator, Seward, Norfolk, England). Serial dilutions of the
suspension were made in BPW and analysed for AMC,
incubated for 2448 h at 30C, and E. coli for 24 h at
44C, according to the standard culture methods (ISO
4833:2003; ISO 16649-2:2001). For the detection of
E. coli O157:H7, 25 g of each sample was diluted in
225 ml of Modified Tryptone Soya broth added with
novobiocin homogenized as previously described and

incubated for 1218 h at 42C, according to the standard culture method (ISO 16 654:2001). Twenty-five
grams of each sample was diluted in 225 ml of BPW
and homogenized as previously described, incubated for
18 h at 37C for the detection of Salmonella spp.
Another 25 g of each sample was diluted in 225 ml of
Half-Fraser broth, homogenized and then incubated for
24 h at 30C for the detection of L. monocytogenes,
according to ISO methods (ISO 6579:2002; ISO 112901:1996).
Molecular method
Escherichia coli O157:H7, Salmonella spp. and L. monocytogenes were detected by PCR Bax System (DuPontQualicon, Geneva, Switzerland), according to the
manufacturers instructions. Five microliters of the second
enrichment broth (EC-Broth) for the detection of E. coli
O157:H7; 5 ll of the second enrichment broth (Brain
Heart Infusion) for the detection of Salmonella spp and
5 ll of the second enrichment broth (MOPS-BLEB) for
the detection of L. monocytogenes were added to 200 ll of
the proteinase containing lysis buffer provided with the
BAX kits (Du Pont-Qualicon). Samples were incubated
for 20 min at 37C, for E. coli O157:H7 and Salmonella
spp, and for 1 h at 55C, for L. monocytogenes. After
incubation, all samples were boiled for 10 min and then
cooled on ice. Fifty microliters of the lysate was added to
the PCR reagent tablet that included an internal amplification control (IAC). Finally, to determine the
positive results of E. coli O157:H7, Salmonella spp and
L. monocytogenes, the amplified DNAs were automatically

Table 1 Microbiological (bacterial and virological) determinations using cultural and molecular methods
Microbiological determinations

Methods

Description

Aerobic Mesophilic
Count (AMC)

ISO 4833 Ed. 2003

E. coli

ISO 16 649-2 Ed. 2001

E. coli O157

ISO 16 654 Ed. 2001

Salmonella spp

PCR BAX System-DuPont Qualicon

ISO 6579 Ed. 2002

Listeria monocytogenes

PCR BAX System-DuPont Qualicon

ISO 11 290-1 2 Ed. 1996 98

Hepatitis A virus (HAV)

Norovirus (NoV)

PCR BAX System-DuPont Qualicon

RT-nested-PCR
RT-booster-PCR

Food Microbiology and animal feed Horizontal method for the

enumeration of micro-organisms. Colony-count technique
at 30C
Food Microbiology and animal feed Horizontal method for the
enumeration of beta-glucuronidase-positive Escherichia coli.
Part 2: Colony-count technique at 44C using 5-bromo-4chloro-3-indolyl beta-D-glucuronide.
Food Microbiology and animal feed - Horizontal method for the
detection of Escherichia coli O157:H7
PCR Assay for screening E. coli O157:H7
Food Microbiology and animal feed - Horizontal method for
the detection of Salmonella spp
PCR Assay for screening Salmonella spp.
Food Microbiology and animal feed - Horizontal method for the
detection and enumeration of Listeria monocytogenes. Part 1:
Detection method; Part 2: Enumeration method.
PCR Assay for screening Listeria monocytogenes
De Medici et al. (2001); Croci et al. (2007).
De Medici et al. (2004); Croci et al. (2007).

998

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M. De Giusti et al.

analysed by fluorescent signals generated by the specific

amplicons.
Virological determination
The concentration of the viral particles of HAV and NoV
was performed as described by Rzezutka et al. (2005).
Ten grams of whole vegetables was placed in a 250-ml
centrifuge tube (Beckman Coulter, Cassina De Pecchi,
Italy). Twenty millilitres of a solution containing
1 mol l)1 sodium bicarbonate containing 1% (w v) soya
protein powder (90% Protein Powder, Holland and
Barrett, Nuneaton, UK) was added to the vegetables. The
centrifuge tube containing the sample was shaken at
room temperature for 15 min at 500 rev min)1 (Heidolph
Unimax X 2010, Wolf Laboratories Limited, York,
UK) then centrifuged at 8000 g for 3 min at room
temperature (Avanti TM J-20 XP Centrifuge, Beckman
Coulter).
The supernatant was decanted into a clean 250-ml
centrifuge tube, and 1 ml CatFloc TL (Calgon Corp,
Pittsburgh, USA) was added to flocculate food solids, and
200 ll Pectinase (Pectinase from A. Aculeatus, Sigma, St
Louis, MO, USA) was added to remove residual pectin.
The sample was centrifuged at 28 000 g for 30 min at
25C to facilitate the action of pectinase (Avanti TM J-20
XP Centrifuge Beckman Coulter). The supernatant was
ultracentrifuged for 2 h at 235 000 g (Optima LE 80K
Ultracentrifuge, Beckman Coulter, Florence, Italy). The
supernatant was discarded, and the pellet was resuspended in 500 ll phosphate-buffered saline (PBS). The
vegetable extract was either used immediately for
extraction of viral RNA, or stored at )20C until RNA
extraction.
Sample extraction of viral RNA
QIAamp viral RNA mini kit (Qiagen, Valencia, CA,
USA) was used to extract the viral RNA from the vegetable samples at a final volume of 50 ll in accordance with
the manufacturers instructions.
HAV detection using Reverse Transcription (RT)-NestedPCR
RT-nested-PCR was performed according to De Medici
et al. (2001) and Croci et al. (2007).
Five microlitres of viral RNA extract was added to
85 ll of RT reaction mixture containing: 1 PCR Buffer
II (Perkin Elmer, Monza, Milano, Italy), 2 mmol l)1
MgCl2 (Perkin-Elmer, Monza, Milano, Italy), 02
mmol l)1 of each deoxynucleoside triphosphate (dNTP)
(Takara-Shuzo, Japan), 20 U of RNAsin (Promega,
Madison, WI, USA), 125 U of AMV reverse transcriptase
(Promega, Florence, Italy) and 100 pmoles of primer anti-

Italian RTE salads safety

sense (5-CATATGTATGGTATCTCAACAA3). The

reverse transcription conditions were 60 min at 42C and
5 min at 95C. For each reaction set-up, negative and
positive controls (RNA extracted from HAV stock solution) were included.
The first amplification was performed adding 90 ll
cDNA to 10 ll of mixture containing: 100 pmoles of primer sense (5-CAGGGGCATTTAGGTTT-3), 25 U of
Taq DNA polymerase (Perkin Elmer) and DNase-RNasefree water (Sigma, USA).
The thermocycling conditions were 30 cycles for 25 s at
95C, for 10 s at 49C and for 1 min at 70C. A final
extension was performed for 5 min at 72C.
In each reaction of the second amplification (nested
PCR), 5 ll of amplicon was added to 95 ll of mixture
containing: 1 PCR Buffer II (Perkin Elmer), 2 mmol l)1
MgCl2, 02 mmol l)1 of each dNTP, 100 pmoles of
primer sense (5-TGATAGGACTGCAGTGACT-3), 100
pmoles of primer antisense (5-CCAATTTTGCAACTTCATG-3) and 25 U of Taq DNA polymerase (Perkin
Elmer). The amplification conditions used were the same
as those described for the first PCR amplification.
NoV detection using RT-booster-PCR
RT-booster-PCR was performed according to that of De
Medici et al. (2004) and Croci et al. (2007) Reverse
Transcriptase (RT). In each reaction, 5 ll of viral RNA
extract was added to 45 ll of RT reaction mixture containing: 1 AMV Tfl buffer, 1 mmol l)1 MgSO4,
02 mmol l)1 of each dNTP, 5 U of AMV reverse
transcriptase (Access RT-PCR System Promega, Florence,
Italy), 4 ll of 5 lmol l)1 primer JV13 (5-TCATCATCACCATAGAAAGAG-3). The reverse transcription
conditions were 60 min at 48C and 5 min at 99C. For
each reaction set-up, negative and two positive controls
(one for GI and one for GII) were included.
The first amplification was performed by adding to the
reverse transcription a reaction mix containing 1 ll of Tfl
DNA polymerase (5 U ll)1), 1 ll of 5 lmol l)1 primer
JV13, 1 ll of 5 lmol l)1 primer JV12 (5-ATACCACTATGATGCAGATTA-3) and 2 ll of PCR grade
water. Samples were then denatured for 2 min at 94C
and subjected to 20 cycles for 1 min at 94C, for 4 min
at 37C and for 2 min at 68C. A final extension was then
performed for 7 min at 68C. The second amplification
step (booster PCR) was performed by adding 5 ll of the
first PCR product to 45 ll reaction mixture containing:
1 AMV Tfl buffer, 1 mmol l)1 MgSO4, 02 mmol l)1 of
each dNTP, 5 U of Tfl polymerase, primers JV13 and
JV12 1 lmol l)1. Cycling conditions were for 2 min at
94C, followed by 40 cycles for 1 min at 94C, for
15 min at 37C, for 2 min at 68C and a final extension
for 7 min at 68C.

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Italian RTE salads safety

M. De Giusti et al.

Electrophoresis PCR products were run in 2% agarose

gel (Kodak) in the presence of ethidium bromide and
photographed (Bio-Rad Chemi Doc, Paris, France).

Results
Salmonella spp was not detected in any RTE samples using
both cultural and molecular methods. On the contrary,
2 265 (075%) of the whole vegetables samples (rucola
lettuce) were positive for Salmonella, with the molecular
method and in one sample the presence of Salmonella was
also confirmed by the cultural method. Isolated Salmonella strain was serotyped as Salmonella umbilo ser 0:28
according to Kauffman and White (Popoff et al. 2003),
from the Reference Laboratory of Enteric Bacteria at the
National Institute of Health, Rome, Italy.
Three positive samples for L. monocytogenes were
found: 1 265 in whole vegetable samples (rucola lettuce,
161%) and 2 699 (029%) in RTE samples (mixed
lettuce). All PCR L. monocytogenes positive samples were
not confirmed by the cultural method.

Statistical analysis
Prevalence, mean values and standard deviations (SD) of
all variables were calculated. The Wilcoxon test was used
to evaluate the effect of different technologies comparing
the median values at different analytical times [after packaging (T0-T1); at the expiration date (T1-T2) and 2 days
after the expiration date (T2-T3)]. KruskalWallis test was
used to test the equality of medians among the producers
at the time of production. Differences were considered statistically significant when P-values were lower than 005.
All statistical calculations were performed using Stata
ver. 8.0 (College Station, TX, Stata Corporation,Texas,
USA, 2003).
Table 2 Aerobic Mesophilic Count (AMC) results Producer 1

Number and percentage of samples

at the indicated intervals (CFU g)1)
no. (%)
Samples
Whole vegetables
T0
Endive
Curly endive
Rucola lettuce
Valerian
Red chicory
RTE vegetables
T1 (1st day)
Endive
Curly endive
Rucola lettuce
Valerian
Mixed salad
T2 (5th day)
Endive
Curly endive
Rucola lettuce
Valerian
Mixed salad
T3 (7th day)
Endive
Curly endive
Rucola lettuce
Valerian
Mixed salad

No. 460

Mean CFU g)1

SD CFU g)1

Median CFU g)1

Range CFU g)1

115
23
23
23
23
23

12
19
84
12
14
79

109
109
108
109
109
108

33
55
23
31
26
13

109
109
109
109
109
109

15
66
71
15
42
15

108
107
107
108
108
108

20
70
15
24
67
20

10226
10426
10411
10414
10411
10244

115
23
23
23
23
23
115
23
23
23
23
23
115
23
23
23
23
23

25
85
47
11
87
13
91
28
76
13
31
96
11
77
23
27
33
14

108
107
107
108
108
108
109
109
108
109
1010
109
1010
108
109
109
1010
1010

14
22
93
25
32
31
47
96
19
42
97
33
43
20
74
51
81
46

109
108
107
108
109
108
1010
109
109
109
1010
1010
1010
109
109
109
1010
1010

54
32
34
60
13
91
14
98
66
13
29
14
20
68
20
45
32
25

106
106
106
106
107
106
107
106
106
107
107
108
108
107
108
108
108
108

15
21
35
15
46
44
18
43
43
10
17
18
10
10
10
11
18
47

10315
10397
10333
10388
10415
10311
10442
10442
10470
10520
10542
10415
10330
10386
10636
10320
10430
10422

106*

106109

>109

1010
1010
1010
1010
1010
109

21 (183)
4 (174)
4 (174)
4 (174)
4 (174)
5 (217)

69 (60)
13 (565)
15 (652)
14 (609)
14 (609)
13 (565)

25 (217)
6 (261)
4 (174)
5 (217)
5 (217)
5 (217)

1010
108
108
108
1010
109
1011
1010
109
1010
1011
1011
1011
109
1010
1010
1011
1011

42 (365)
10 (435)
10 (435)
5 (217)
7 (304)
10 (435)
26 (226)
4 (174)
5 (2175)
8 (348)
4 (174)
5 (217)
13 (113)
2 (87)
3 (130)
3 (130)
2 (87)
3 (130)

70 (609)
13 (565)
13 (565)
18 (783)
14 (609)
12 (522)
72 (626)
17 (739)
15 (6525)
11 (478)
15 (652)
14 (609)
65 (565)
17 (739)
12 (522)
11 (478)
13 (565)
12 (522)

3 (26)
0 (00)
0 (00)
0 (00)
2 (87)
1 (43)
17 (148)
2 (87)
3 (130)
4 (174)
4 (174)
4 (174)
37 (322)
4 (174)
8 (348)
9 (392)
8 (348)
8 (348)

*1 106 CFU g)1: microbiological limits proposed for whole vegetables (Gelosa 1998); 1 106 CFU g)1: microbiological limits proposed for
RTE vegetables according to the DM French Republic 22 03 93.
1 107 CFU g)1 microbiological limits proposed for RTE vegetables in the PHLS Public Health Laboratory Service (2000); T0: whole vegetables
analysed at the second day after harvesting; T1: RTE vegetables analysed at the first day after packaging; T2: RTE vegetables analysed at the
expiration date according to the producers (the fifth day for Producer 1 and the seventh day for Producer 2 and 3, after packaging); T3: RTE
vegetables analysed 2 days after the expiration date, given by the producers.

1000

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M. De Giusti et al.

Italian RTE salads safety

Escherichia coli O157:H7 was not detected in any of the

964 samples analysed both with cultural and molecular
methods.
HAV and NoV were not detected in any of the 124
samples analysed.
Tables 27 show the results of the enumeration of
AMC and E. coli, reported as mean, standard deviation,
median and distribution of the samples in the defined
range.
The whole vegetables (T0) of the three producers
showed similar AMC median value (median values:
15 108 CFU g)1, 14 108 CFU g)1, 77 108 CFU g)1,
respectively); however AMC count of Producer 1 started
at 200 102 CFU g)1 in red chicory and increased to
26 1010 CFU g)1 in endive. Conversely, Producer 2 and
3 showed minimum values (16 107 CFU g)1 of AMC
in curly endive) higher than Producer 1 (19
106 CFU g)1 in rucola lettuce). Curly endive was consis-

tently
found
to
have
maximum
values
of
14 1012 CFU g)1 (Producer 2) and 80 1010 CFU g)1
(Producer 3), as shown in Tables 3 and 4. Mean values of
AMC of whole vegetables for Producer 1 showed a statistical difference when compared to those of Producers 2 and
3 (P < 005). Producer 1, also had 183% of all its whole
vegetables samples within the limit (1 106 CFU g)1)
for the mean values of AMC (Table 2), as previously proposed by other authors (Gelosa 1998); however, mean
values of AMC for Producer 2 and 3 were higher than this
limit (Tables 3 and 4). AMC mean values detected in vegetables processed by Producer 1 were lower than 1 decimal
logarithm both in whole and in RTE vegetables, with a
statistically
significant
decrease
after
processing
(P < 0001).
After processing, the RTE vegetables at the beginning
of their shelf life (T1) showed AMC median values
lower by about two decimal logarithm for Producers 1,

Table 3 Aerobic Mesophilic Count (AMC) results Producer 2

Number and percentage of
samples at the indicated intervals
(CFU g)1) no. (%)
Samples
Whole vegetables
T0
Endive
Curly endive
Red chicory
Rucola lettuce
RTE vegetables
T1 (1st day)
Endive
Curly endive
Red chicory
Rucola lettuce
Mixed salad
T2 (7th day)
Endive
Curly endive
Red chicory
Rucola lettuce
Mixed salad
T3 (9th day)
Endive
Curly endive
Rucola lettuce
Red chicory
Mixed salad

No. 312

Mean CFU g)1

SD CFU g)1

78
23
11
17
27

43
46
19
91
40

1010
108
1011
109
1010

17
31
43
11
77

1011
108
1011
1010
1010

14
46
29
37
35

108
108
109
109
1010

16
20
16
47
20

78
23
11
4
27
13
78
23
11
4
27
13
78
23
11
27
4
13

12
20
62
37
52
55
74
28
32
66
85
14
34
87
58
24
25
55

109
108
109
106
108
108
108
108
107
109
108
108
109
108
108
109
1010
109

77 109
23 108
21 1010
10 106
54 108
18 108
16 109
41 108
46 107
34 109
83 108
16 108
86 109
10 109
70 108
45 109
25 1010
998 109

15
99
50
33
18
60
19
16
16
67
72
53
48
46
30
12
17
20

107
106
106
106
108
106
108
108
107
109
108
106
108
108
108
109
1010
108

<1068 1010
28 10446
<1068 1010
18 10665
34 10512
40 10551
28 10410
24 10515
28 10415
30 10910
21 10633
64 10638
27 10761
10 10943
27 10724
90 10719
37 10961
49 10726

Median CFU g)1

Range CFU g)1

10714
10715
10714
10737
10734

106

1012
109
1012
1010
1011

108
106
109
109
1010
109
108
1010
109
108
1010
109
109
1010
1010
1010

106109

>109

0 (00)
0 (00)
0 (00)
0 (00)
0 (00)

36 (462)
22 (957)
3 (273)
3 (176)
8 (296)

42 (538)
1 (43)
8 (727)
14 (824)
19 (704)

12 (154)
4 (174)
4 (364)
0 (00)
3 (111)
1 (77)
9 (115)
5 (217)
3 (273)
0 (00)
0 (00)
1 (77)
0 (00)
0 (00)
0 (00)
0 (00)
0 (00)
0 (00)

54 (692)
19 (926)
6 (545)
4 (1000)
14 (519)
11 (846)
52 (667)
16 (696)
8 (727)
0 (00)
16 (593)
12 (923)
47 (603)
17 (739)
9 (818)
12 (444)
0 (00)
9 (692)

12 (154)
0 (00)
1 (91)
0 (00)
10 (370)
1 (77)
17 (218)
2 (87)
0 (00)
4 (1000)
11 (407)
0 (00)
31 (397)
6 (261)
2 (182)
15 (556)
4 (1000)
4 (307)

*1 106 CFU g)1: microbiological limits proposed for whole vegetables (Gelosa 1998); 1 106 CFU g)1: microbiological limits proposed for
RTE vegetables according to the DM French Republic 22 03 93.
1 107 CFU g)1 microbiological limits proposed for RTE vegetables in the PHLS Public Health Laboratory Service (2000); T0: whole vegetables
analysed at the second day after harvesting; T1: RTE vegetables analysed at the first day after packaging; T2: RTE vegetables analysed at the
expiration date according to the producers (the fifth day for Producer 1 and the seventh day for Producer 2 and 3, after packaging); T3: RTE
vegetables analysed 2 days after the expiration date, given by the producers.
2010 The Authors
Journal compilation 2010 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 9961006

1001

Italian RTE salads safety

M. De Giusti et al.

while median values of Producers 2 and 3 decreased by

about 1 decimal logarithm compared to AMC found in
whole vegetables (T1 median values: 54 106 CFU g)1,
15 107 CFU g)1, 37 107 CFU g)1, respectively, for
Producers 1, 2 and 3). The prevalence of samples of RTE
vegetables at T1 that showed values of AMC below the
limits required by the international standard, MD 22
mars (1993) (1 106 CFU g)1), and those reported in
the PHLS Guidelines 2000 (1 107 CFU g)1) was
365% (Producer 1), 154% (Producer 2) and 175%
(Producer 3). The difference among AMC median valued
at T1 was statistically significant (P = 0011). The AMC,
shown in Tables 24, ranged from 15 103 CFU g)1 in
rucola lettuce to 15 1010 CFU g)1 in valerian (T1,
Producer 1), from <10 to 68 1010 CFU g)1 in curly
endive (T1, Producer 2) and from 15 104 CFU g)1 in
rucola lettuce to 81 108 CFU g)1 in a mix of lettuces (T1, Producer 3). The decrease in AMC values
between T0 and T1 was statistically significant only for
RTE salads of Producers 1 and 2 (P < 0001 in both
cases).

At the end of shelf life (T2), there was a statistically

significant increase in AMC values in all the RTE salads
of the 3 Producers. However, the RTE salads of Producer 3
showed a greater increase in AMC values than Producer 1
and 2 (T2 median values: 14 107 CFU g)1,
19 108 CFU g)1, 12 109 CFU g)1, respectively for
Producers 1, 2 and 3).
Two days after the expiration date (T3), RTE vegetables
again had a statistically significant increase in all AMC
values in the 3 production processes (Table 24).
As shown in Tables 57, Producer 1 showed a higher
frequency of E. coli absence in whole vegetables and RTE
vegetables (757 and 5739%, respectively), followed by
Producer 2 (3974 and 2820%, respectively) and Producer
3 (1154 and 25%, respectively).
Escherichia coli median values were low for all three
producers; however, the values started at <10 CFU g)1 in
all vegetables (Producer 1, T0) and increased to
11 103 CFU g)1 (Producer 3, T1). Unlike Producer 2
and 3 that had the highest values throughout the whole
production chain.

Table 4 Aerobic Mesophilic Count (AMC) results Producer 3

Number and percentage of
samples at the indicated intervals
(CFU g)1) no. (%)
Samples
Whole vegetables
T0
Endive
Curly endive
Rucola lettuce
Red chicory
Red lattuce
RTE vegetables
T1 (1st day)
Rucola letture
Mixed lettuce
Mixed salad
T2 (7th day)
Rucola lettuce
Mixed lettuce
Mixed salad
T3 (9th day)
Rucola lettuce
Mixed lettuce
Mixed salad

No. 192

Mean CFU g)1

SD CFU g)1

MedianCFU g)1

RangeCFU g)1

72
22
22
12
10
6

37
24
86
12
19
51

109
109
109
108
109
108

10
41
17
26
43
39

1010
109
1010
108
109
108

77
10
44
41
44
48

108
109
109
106
108
108

19
10
30
19
13
24

10680
10820
10880
10672
10714
10712

1010
1010
1010
108
1010
109

40
12
6
22
40
12
6
22
40
12
6
22

11
10
32
61
60
23
34
26
25
25
63
26

108
108
108
107
1010
1010
1011
109
1011
1010
1011
1011

18
12
35
10
32
47
81
44
84
49
15
82

108
108
108
108
1011
1010
1011
109
1011
1010
1012
1011

37
39
22
37
12
95
95
35
63
65
17
76

107
107
108
107
109
108
109
108
108
108
108
108

15
15
14
70
50
50
50
45
18
22
80
18

10481
10435
10781
10547
10520
10515
10920
10618
10638
10613
10738
10638

108
108
108
108
1012
1011
1012
1010
1012
1011
1012
1012

106*

106109

>109

0 (00)
0 (00)
0 (00)
0 (00)
0 (00)
0 (00)

42 (583)
10 (455)
8 (364)
12 (1000)
7 (700)
5 (833)

30 (417)
12 (545)
14 (636)
0 (00)
3 (300)
1 (167)

7 (175)
1 (83)
0 (00)
6 (273)
1 (25)
1 (83)
0 (00)
0 (00)
0 (00)
0 (00)
0 (00)
0 (00)

33 (825)
11 (917)
6 (1000)
16 (727)
18 (450)
5 (417)
0 (00)
13 (591)
24 (600)
7 (583)
4 (667)
13 (591)

0 (00)
0 (00)
0 (00)
0 (00)
21 (525)
6 (500)
6 (1000)
9 (409)
16 (400)
5 (417)
2 (333)
9 (409)

*1 106 CFU g)1: microbiological limits proposed for whole vegetables (Gelosa 1998); 1 106 CFU g)1: microbiological limits proposed for
RTE vegetables according to the DM French Republic 22 03 93.
1 107 CFU g)1 microbiological limits proposed for RTE vegetables in the PHLS Public Health Laboratory Service (2000); T0: whole vegetables
analysed at the second day after harvesting; T1: RTE vegetables analysed at the first day after packaging; T2: RTE vegetables analysed at the
expiration date according to the producers (the fifth day for Producer 1 and the seventh day for Producer 2 and ,3 after packaging); T3: RTE
vegetables analysed 2 days after the expiration date, given by the producers.

1002

2010 The Authors

Journal compilation 2010 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 9961006

M. De Giusti et al.

Italian RTE salads safety

At the beginning of shelf life, processed vegetables

showed a statistical increase in E. coli concentration for
both Producer 1 and Producer 3 (being more pronounced
in Producer 3), and a slight decrease, which was not statistically significant in Producer 2.
At the end of shelf life (T2), there was a no statistically
significant increase in E.coli values in RTE vegetables of
Producers 1 and 2. Instead, a statistically significant
increase was found for Producer 3 (P = 0045).
Two days after the expiration date (T3), RTE vegetables
showed a statistically significant increase in E. coli values
both for Producer 1 and Producer 2, but were not statistically significant for Producer 3 (Table 57).

Discussion
All the results of the present study were within the
limits stipulated by the EC Regulation 1441 2007 CE
during the investigation into the different technologies
in terms of microbial quality and safety of RTE vegetables.
Only 2 of the 699 RTE samples analysed were positive
for L. monocytogenes (029%) using the PCR method, but
these results were not confirmed by the cultural reference
methods. The results underline the necessity to confirm
the PCR positive results with the cultural methods in
evaluating of food safety in vegetables.

Table 5 Escherichia coli results Producer 1

Samples

No.
460

Mean
CFU g)1

Whole vegetables
T0
Endive
Curly endive
Rucola lettuce
Valerian
Red chicory

115
23
23
23
23
23

75
96
68
17
72
35

RTE vegetables
T1 (1st day)
Endive
Curly endive
Rucola lettuce
Valerian
Mixed salad
T2 (5th day)
Endive
Curly endive
Rucola lettuce
Valerian
Mixed salad
T3 (7th day)
Endive
Curly endive
Rucola lettuce
valerian
Mixed salad

115
23
23
23
23
23
115
23
23
23
23
23
115
23
23
23
23
23

67
12
10
39
93
10
46
13
53
56
23
21
66
19
18
15
31
12

101
101
101
102

101

102
103
103
101

103
103
103
102
101

104
103
103
104
103
101
104

SD CFU g)1

25
32
23
38
31
10

25
32
31
11
43
32
43
32
23
21
78
96
37
52
65
51
14
50

102
102
102
102
101
102

103
103
103
102
101
103
104
103
103
102

104
104
103
104
103
102
104

Median
CFU g)1

Range
CFU g)1

<10
<10
<10
<10
<10
<10

<1011
<1011
<1011
<1011
<1015
<1043

<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
23 101
<10
<10
<10
75 101

<1011
<1011
<1011
<1046
<1021
<1011
<1046
<1011
<1011
<1010
<1036
<1046
<1030
<1023
<1030
<1024
<1067
<1024

Number and percentage of samples at the indicated

intervals (CFU g)1) no. (%)
102

103
103
103
103
102
102

104
104
104
102
102
104
105
104
104
103
101
105
105
104
105
104
102
105

102103

102 (887)
21 (913)
20 (870)
19 (8265)
22 (957)
20 (870)

7
0
2
1
1
3

>103

(61)
(00)
(87)
(435)
(43)
(130)

6 (52)
2 (87)
1 (43)
3 (130)
0 (00)
0 (00)

Satisfactory
102

Unsatisfactory
> 102

103

>103

95 (826)
17 (739)
19 (826)
20 (870)
22 (957)
17 (739)
94 (817)
15 (652)
19 (826)
21 (913)
23 (1000)
16 (696)
84 (730)
16 (696)
17 (739)
16 (696)
22 (957)
13 (565)

20 (174)
6 (261)
4 (174)
3 (130)
1 (43)
6 (261)
21 (183)
8 (348)
4 (174)
2 (87)
0 (00)
7 (304)
31 (270)
7 (304)
6 (261)
7 (304)
1 (43)
10 (435)

105 (913)
19 (826)
19 (826)
23 (1000)
23 (1000)
21 (913)
103 (896)
17 (739)
22 (957)
23 (1000)
23 (1000)
18 (783)
96 (835)
18 (783)
18 (783)
19 (826)
23 (1000)
18 (783)

10 (87)
4 (174)
4 (174)
0 (00)
0 (00)
2 (87)
12 (104)
6 (261)
1 (43)
0 (00)
0 (00)
5 (217)
19 (165)
5 (217)
5 (217)
4 (174)
0 (00)
5 (217)

*1 103 CFU g)1: microbiological limits proposed for whole vegetables (Gelosa L 1998Gelosa 1998).
1 102 CFU g)1microbiological limits proposed for RTE vegetables according to the DM French Republic 22 03 93 and the PHLS Public
Health Laboratory Service 2000.
1 103 CFU g)1: official foodstuff microbiological criteria for RTE vegetables at the producer according to the EC Regulation no. 1441 2007;
T0: whole vegetables analysed at the second day after harvesting; T1: RTE vegetables analysed at the first day after packaging; T2: RTE vegetables
analysed at the expiration date according to the producers (the fifth day for Producer 1 and the seventh day for Producer 2 and 3, after
packaging); T3: RTE vegetables analysed 2 days after the expiration date, given by the producers.

2010 The Authors

Journal compilation 2010 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 9961006

1003

Italian RTE salads safety

M. De Giusti et al.

Table 6 Escherichia coli results Producer 2

Number and percentage of samples at the indicated interval
(CFU g)1) no. (%)
Samples

No.
312

Mean
CFU g)1

Whole vegetables
T0
Endive
Curly endive
Red chicory
Rucola lettuce

78
23
11
17
27

4.2
1.6
1.1
1.9
5.7

RTE vegetables
T1 (1st day)
Endive
Curly endive
Red chicory
Rucola lettuce
Mixed salad
T2 (7th day)
Endive
Curly endive
Red chicory
Rucola lettuce
Mixed salad
T3 (9th day)
Endive
Curly endive
Red chicory
Rucola lettuce
Mixed salad

78
23
11
4
27
13
78
23
11
4
27
13
78
23
11
4
27
13

6.3
6.7
1.1
1.5
1.6
2.7
3.1
1.2
3.1
0.0
5.5
4.9
1.5
1.3
1.5
9.0
7.2
7.0

104
103
102
105
102

103
102
102
102
104
103
103
103
102

103
103
104
103
103
101
103
104

SD CFU g)1

3.6
5.0
3.3
7.8
1.6

4.9
1.7
1.9
2.1
8.3
4.7
1.3
2.3
7.0
1.5
2.1
8.2
7.6
2.0
2.3
1.0
2.2
1.8

105
103
102
105
103

104
103
102
102
104
103
104
103
102

104
103
104
103
103
102
104
105

Median
CFU g)1

Range
CFU g)1

1.8 101
7.4 101
<10
<10
1.1 102

<103.2
<102.4
<101.1
<103.2
<108.3

4.4
5.5
3.0
6.4
2.3
2.3
1.5
2.0
0.0
0.0
1.0
0.0
7.3
4.3
1.1
4.9
1.0
2.1

101
101
101
101
102
101

101
101
101
103
101
101
103

106
104
103
106
103

<104.3 105
<107.7 103
<104.6 102
6.4 1014.6 102
<104.3 105
<101.1 104
<101.1 105
<109.3 103
<102.3 103
<10
<101.1 105
<102.4 104
<106.4 105
<106.0 103
<107.5 103
2.3 1012.4 102
<109.2 104
<106.4 105

102

102103

>103*

51
15
10
13
13

12 (15.4)
1 (4.3)
0 (0.0)
1 (5.9)
10 (37.0)

15 (19.2)
7 (30.5)
1 (9.1)
3 (17.6)
4 (14.8)

(65.4)
(65.2)
(90.9)
(76.5)
(48.2)

 Satisfactory
102

 Unsatisfactory
>102

 103

 >103

45 (57.7)
13 (56.5)
7 (63.6)
3 (75.0)
17(63.0)
4 (30.8)
49 62.8)
13 (56.5)
8 (72.7)
4 (100.0)
17 (63.0)
7 (53.8)
38 48.7)
12 (52.2)
4 (36.4)
3 (75.0)
17 (63.0)
5 (38.5)

33 (42.3)
10 (43.5)
4 (36.4)
1 (25.0)
10 (37.0)
9 (69.2)
29 (37.2)
10 (43.5)
3 (27.3)
0 (0.0)
10 (37.0)
6 (46.2)
40 (51.3)
11 (47.8)
7 (63.6)
1 (25.0)
10 (37.0)
8 (61.5)

66 (84.6)
19 (82.6)
11 (100.0)
4 (100.0)
23 (85.2)
9 (69.2)
62 (79.5)
17 (73.9)
10 (90.9)
4 (100.0)
22 (81.5)
9 (69.2)
46 (59.0)
15 (65.2)
5 (45.5)
4 (100.0)
17 (63.0)
5 (38.5)

12 (15.4)
4 (17.4)
0 (0.0)
0 (0.0)
4 (14.8)
4 (30.8)
16 (20.5)
6 (26.1)
1 (9.1)
0 (0.0)
5 (18.5)
4 (30.8)
32 (41.0)
8 (34.8)
6 (54.5)
0 (0.0)
10 (37.0)
8 (61.5)

*1 103 CFU g)1: microbiological limits proposed for whole vegetables (Gelosa L, 1998).
1 102 CFU g)1microbiological limits proposed for RTE vegetables according to the DM French Republic 22 03 93 and the PHLS Guidelines
2000.
1 103 CFU g)1: official foodstuff microbiological criteria for RTE vegetables at the producer according to the EC Regulation no. 1441 2007;
T0: whole vegetables analysed at the second day after harvesting; T1: RTE vegetables analysed at the first day after packaging; T2: RTE vegetables
analysed at the expiration date according to the producers (the fifth day for Producer 1 and the seventh day for Producer 2 and 3, after
packaging); T3: RTE vegetables analysed 2 days after the expiration date, given by the producers.

Many studies have reported outbreaks related to the

consumption of vegetables even if the foodborne pathogens were not found in the RTE samples.
Surprisingly, there was an absence of Listeria spp and a
low prevalence of Salmonella spp (1 265, 0004%) in
whole vegetables when using the cultural method. Even if,
PCR positive Listeria and Salmonella had been considered,
the prevalence of these micro-organisms (0004% and
075%, respectively) are lower than those reported by
other authors (Gola et al. 1990; Breer and Baumgartner
1992; MacCowan et al. 1994).
This study confirmed the results of other recent studies
regarding RTE vegetable safety, as E. coli O157:H7 was
not detected in samples analysed (Sagoo et al. 2003;
Froder et al. 2007; Abadias et al. 2008). Nevertheless, an
outbreak of gastroenteric disease because of E. coli
O157:H7 was described in the USA after RTE vegetable
consumption (Ackers et al. 1998).
It is important that precautions should be taken in all
production steps, as some pathogenic bacteria (Listeria
1004

spp) are able to survive and grow on vegetables stored at

refrigeration temperature (Abdul-Raouf et al. 1993).
Leafy vegetables have a high viral capacity of adsorption and therefore the most favourable conditions for
viral persistence. HAV can be recovered from lettuce
almost 9 days after contamination and washing apparently does not guarantee the elimination of HAV from
the leaf surface (Croci et al. 2002).
Although AMC does not define the microbiological
safety of the products, it is still a very useful tool to monitor the effect of different technologies on the microbiological quality of the products. Also, the determination of
AMC should be used as a simple and inexpensive parameter to control the effectiveness of the process in the HACCP monitoring plan.
It is interesting to note that the prevalence of samples
of RTE vegetables that showed values of AMC below the
limits required by the international MD 22 mars (1993)
(1 106 CFU g)1) standards and those reported in the
PHLS Guidelines, 2000 (1 107 CFU g)1) was 365%

2010 The Authors

Journal compilation 2010 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 9961006

M. De Giusti et al.

Italian RTE salads safety

Table 7 Escherichia coli results Producer 3

Samples

No.
192

Mean
CFU g)1

Whole vegetables
T0
Endive
Curly endive
Rucola lettuce
Red chicory
Red lettuce

72
22
22
12
10
6

72
55
15
35
63
23

102
102
103
102
101
102

SD
CFU g)1

22
11
37
66
47
43

103
103
103
102
101
102

Median
CFU g)1

68
80
20
12
74
23

101
101
101
102
101
101

Number and percentage of samples at the

indicated intervals (CFU g)1) no. (%)

Range
CFU g)1

<1016 104
<1050 103
<1016 104
<1024 103
<1012 102
9011 103

102

102103

>103

46 (639)
14 (636)
15 (682)
6 (500)
7 (700)
4 (666)

14 (194)
3 (137)
2 (91)
5 (417)
3 (300)
1 (167)

12 (167)
5 (227)
5 (227)
1 (83)
0 (00)
1 (167)

Satisfactory
102
RTE vegetables
T1 (1st day)
Rucola lettuce
Mixed lettuce
Mixed salad
T2 (7th day)
Rucola lettuce
Mixed lettuce
Mixed salad
T3 (9th day)
Rucola lettuce
Mixed lettuce
Mixed salad

40
12
6
22
40
12
6
22
40
12
6
22

38
90
13
17
31
98
34
51
21
41
38
16

103
103
103
103
105
104
105
105
105
105
103
105

77
12
17
25
17
14
58
23
60
86
37
49

103
104
103
103
106
105
104
106
105
105
103
105

11
46
78
46
66
32
15
13
36
26
33
28

103
103
102
102
102
104
104
103
103
104
103
103

<1039 104
15 10139
<1046 103
17 10111
<1011 107
<1046 105
92 10215
<1011 107
<1024 106
<1024 106
74 10211
<1021 106

104
104

105

104

6 (150)
2 (167)
1 (167)
3 (136)
6 (150)
2 (167)
0 (00)
4 (182)
6 (150)
1 (83)
0 (000)
5 (227)

Unsatisfactory
> 102

103

>103

34 (850)
10 (833)
5 (833)
19 (864)
34 (850)
10 (833)
6 (1000)
18 (818)
34 (850)
11 (917)
6 (1000)
17 (773)

18 (450)
3 (250)
3 (500)
12 (545)
14 (350)
4 (333)
1 (167)
9 (409)
11 (275)
1 (83)
2 (333)
8 (364)

22 (550)
9 (750)
3 (500)
10 (455)
26 (650)
8 (667)
5 (833)
13 (591)
29 (725)
11 (917)
4 (667)
14 (636)

1 103 CFU g)1: microbiological limits proposed for whole vegetables (Gelosa 1998).
1 102 CFU g)1 microbiological limits proposed for RTE vegetables according to the DM French Republic 22 03 93 and the PHLS Public
Health Laboratory Service 2000.
1 103 CFU g)1: official foodstuff microbiological criteria for RTE vegetables at the producer according to the EC Regulation no. 1441 2007;
T0: whole vegetables analysed at the second day after harvesting; T1: RTE vegetables analysed at the first day after packaging; T2: RTE vegetables
analysed at the expiration date according to the producers (the fifth day for Producer 1 and the seventh day for Producer 2 and 3, after packaging); T3: RTE vegetables analysed 2 days after the expiration date, given by the producers.
*

(Producer 1), 154% (Producer 2) and 175% (Producer 3).

This prevalence provides an indication of how a rigorous
application of GAP, GMP and HACCP strategies could
attain an optimum vegetable RTE microbiological quality;
however, the products analysed in this study had better
results than those reported by other authors, who found a
prevalence of about 119% for samples within the limit of
1 107 CFU g)1 (Abadias et al. 2008).
The best strategy evidenced, was that of Producer 1,
confirmed by the low contamination of E. coli in whole
vegetables and by the subsequent statistically significant
decrease (P = 0004), after RTE processing.
With respect to the limit established by EC Regulation
1441 2007 (1000 CFU g)1 of E. coli), it was found that
Producer 3 had the highest limit excesses (55% of RTE
analysed), followed by Producer 2 (154%) and Producer 1
(87%). The performance of Producers 1 and 2 confirms
the findings (114%) of Abadias et al. (2008).

The study highlights the need to implement

good hygiene practices to prevent contamination and
bacterial growth in RTE vegetables. The findings also
suggest that methodical precautions implemented in
primary production (GAP) can improve the microbiological quality of whole vegetables, in combination
with the proper application of GMP and HACCP
(even in the absence of chemical antimicrobial treatment or physical microbial stabilization) to obtain a
final product that fully meets the required reference
standards.
Acknowledgements
This research was granted by Italian Government
[Forestry Food Agricultural Ministry (MIPAAF), Food
Quality Research Project (D.M. 591 7303 02, December
23, 2002)]. The authors thank the producers for their

2010 The Authors

Journal compilation 2010 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 9961006

1005

Italian RTE salads safety

M. De Giusti et al.

collaboration in this study and to Maurice Di Santolo for

the linguistic revision of this manuscript.
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Journal compilation 2010 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 9961006