Comparative characterization of Listeria monocytogenes isolated from Portuguese farmhouse ewe's...

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International Journal of Food Micro

Comparative characterization of Listeria monocytogenes isolated from

Portuguese farmhouse ewe’s cheese and from humans

Pedro Leite a, Rui Rodrigues a, MASS Ferreira a, Graca Ribeiro b,

Christine Jacquet c, Paul Martin c, Luisa Brito a,*

a Laboratorio de Microbiologia, Instituto Superior de Agronomia, Tapada da Ajuda 1349-017 Lisbon, Portugalb Servico de Patologia Clınica, Laboratorio de Microbiologia, Hospitais da Universidade de Coimbra, 3000 Coimbra, Portugalc Laboratoire des Listeria, Centre National de Reference des Listeria, WHO Collaborating Center for Foodborne Listeriosis,

Institut Pasteur, 75724 Paris Cedex 15, France

Received 30 July 2004; received in revised form 10 January 2005; accepted 21 May 2005

Abstract

In order to investigate the possible relationships between Listeria monocytogenes strains isolated from farmhouse ewe’s cheese and clinical

strains collected, in partially overlapping dates, from the same geographical area in Portugal, a total of 109 isolates from seven ewe’s cheese

manufactures (n =94) and from humans (n =15) were characterized by serotyping, RAPD, PFGE and allelic analysis of the virulent actA gene.

Serotyping indicated the presence of four different serovars: 1/2a, 1/2b, 1/2c and 4b. The 15 clinical isolates were either serovar 4b (86.7%) or

serovar 1/2b (13.3%). Among the 94 isolates from cheese and related environments the serovars prevalence was 1/2a (1.1%), 1/2b (17.0%), 1/2c

(12.8%) and, unexpectedly, 4b (69.1%). Based on results obtained with PFGE typing of the strains, 25 genotypes were identified, 10 from

farmhouses and 15 from human cases. Isolates from serovars 1/2a and 1/2c were assigned to single genotypes, respectively. Within serovars 1/2b

and 4b three and 20 genotypes were established, respectively. RAPD typing of the isolates rendered 18 types indicating the lack of accuracy of the

primers used in strain differentiation within serovar 4b. The actA gene typing of the strains showed a prevalence of actA gene type I (90.4%)

compared with the rest of the strains that were all actA gene type II (9.6%). In spite of the fact that all the farmhouses were completely

independent, the distribution of L. monocytogenes genotypes, intra and inter cheese manufactures, was relatively homogeneous, suggesting the

existence of resident strains. In contrast, among human isolates there was a great genetic diversity. There was no common genotype between L.

monocytogenes implicated in the cases of listeriosis and these cheese-related isolates, suggesting the absence of a causal relationship.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Listeria monocytogenes; Farmhouse ewe’s cheese; Human listeriosis; Serovar distribution; Molecular typing; actA polymorphism

1. Introduction

Listeria monocytogenes is a human food-borne pathogen

responsible for gastroenteritis, and more severe manifestations

including septicemia, central nervous system infections, and

materno-fetal infections leading to stillbirths and abortions.

Although rare, listeriosis is of public health concern because of

its high case-fatality (20–30%) and the potential of L.

monocytogenes to cause large outbreaks targeting predomi-

nantly pregnant women and immunodeficient individuals. The

0168-1605/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.ijfoodmicro.2005.05.017

* Corresponding author. Tel.: +351 21 365 3240, +351 21 365 3435; fax:

+351 21 365 3238.

E-mail address: [email protected] (L. Brito).

implication of a variety of food products, mostly of dairy and

meat origin, in outbreaks and sporadic cases, and the resulting

increase in product recalls have led to a serious economic

problem associated with L. monocytogenes. Dairy products,

cheese in particular, have been associated with food-borne

listeriosis. In 1985, at least 142 listeriosis cases in California

(USA), including 48 deaths, were linked to Mexican-style

cheese contaminated with this bacterium (Linnan et al., 1988).

An outbreak in Switzerland between 1983 and 1987, involving

at least 122 cases, including 34 deaths, was reported to be due

to contamination of Vacherin Mont d’Or cheese (Bille, 1990).

In 1995, there were 36 cases of listeriosis in France which were

linked to Brie de Meaux, a soft cheese made from raw milk

(Goulet et al., 1995). In this case, no deaths were recorded.

biology 106 (2006) 111 – 121

www.elsevi

P. Leite et al. / International Journal of Food Microbiology 106 (2006) 111–121112

Listeria is widely disseminated in the rural environment

and, consequently, may contaminate milk and production

plants. Pritchard and Donnelly (1999), in a study of dairy

processing facilities, found a significantly higher incidence of

Listeria contamination whenever the farms were contiguous to

the processing facilities, which is the main rule in Portuguese

ewe dairy farms. Domesticated ruminants probably play a key

role in the maintenance of Listeria spp. in the rural

environment via a continuous fecal–oral enrichment cycle

(Vazquez-Boland et al., 2001). Whenever dairy cattle are fed

with ensiled forages, the risk of ruminant listeriosis rises due to

the presence of the pathogen in poorly fermented feeds

(Donelly, 2001). In this case, the main clinical manifestations

of the infection are encephalitis, septicemia and abortion

(Vazquez-Boland et al., 2001) and, usually, the infected

animals die within days. However, L. monocytogenes may

also be shed in milk from mastitic animals. The association of

L. monocytogenes with sub-clinical mastitis in sheep has been

reported, although the reports on these cases are meagre,

especially concerning sheep (Fthenakis et al., 1998; Schoder et

al., 2003). In addition to the quality of silage, other hygiene

parameters, ensured by a good herd health management, have

been identified as essential to the microbiological quality of the

milk (Sanaa et al., 1993; Regli, 2004).

Because of its versatility, Listeria is able to persist in the

environment. Blackman and Frank (1996) demonstrated that L.

monocytogenes was able to form biofilms on contact surfaces

of different food products. These authors concluded that food

residues on wet surfaces, in plant environment, could facilitate

biofilm formation, potentially leading to the spread of this

pathogen throughout the processing plant. Soft cheese is

maturated and further stored at refrigeration temperatures,

situations that favour the survival and growth of L. mono-

cytogenes. For this, it is likely that cheese contaminated with L.

monocytogenes may reach the consumers.

Presently, in Portugal, there is no surveillance for L.

monocytogenes infections and, consequently, there is no

reported human listeriosis associated with food consumption.

From a survey performed on 24 ewe’s cheese manufactures

from the central part of Portugal, from March 2002 to June

2003, 94 isolates were selected for a comparative character-

ization with 15 human isolates, collected between July 1997

and February 2003, from cases of listeriosis, in a public

Hospital from the same region. No relation was previously

reported between the clinical isolates and the ingestion of any

kind of contaminated food. In order to differentiate the isolates,

serotyping, RAPD and PFGE typing and the analysis of the

actA gene polymorphisms were performed. Although serotyp-

ing is less discriminatory than PFGE or RAPD, it is a universal

technique that has been used, for the characterization of L.

monocytogenes, in epidemiological studies, even before the

implementation of molecular techniques and before the

combination of results from serotyping and from molecular

typing (Fleming et al., 1985). To look for specific virulence

markers, the genetic characterization of L. monocytogenes

strains often makes use of the analysis of virulent gene

polymorphisms, in combination with the analysis of the clinical

or food origin of the isolates. The virulent gene actA codes for

the bacterial surface protein ActA that mediate the intra- and

inter-cellular bacteria actin-based movement, and the escape

from the defence mechanisms of the host cells (Kocks et al.,

1992). Previous studies (Wiedmann et al., 1997; Inoue et al.,

2001) had reported the differentiation of two alleles of gene

actA in L. monocytogenes, but the frequencies of these alleles,

among the three lineages, were not completely in accordance.

The present study aimed to characterize L. monocytogenes

cheese and clinical isolates collected, in partially overlapping

dates, from the same geographical area, and to investigate their

potential relationships.

2. Materials and methods

2.1. Farmhouse presentation

During 15 months (that included two consecutive cheese-

making seasons), 24 ewe cheese manufactures, from the central

part of Portugal, were screened for the presence of L.

monocytogenes. All the farmhouses had their own flocks and

their own cheese manufactures. The sheep were only housed

indoors at night, the rest of the time they were on pasture. In

this region, and depending on the season, sheep are fed with

pasture, hay and compound feed. Seventeen of these farm-

houses had flocks with more than 80 sheep, and the other seven

farmhouses corresponded to small flocks (less than 80 sheep).

Depending on the farmhouse, milking was done either

manually or mechanically. To our knowledge, there were no

incidents of listeriosis in the flocks, during the surveyed period.

The 24 farmhouses corresponded to about 10% of all the

producers from this cheese-producing region.

2.2. Collection of samples from farmhouse manufactures

Between March 2002 and June 2003, 113 cheese (weight

ranging from 500 g to 1500 g) from 51 different batches were

collected from 24 ewe’s cheese manufactures, at the central

part of Portugal. The semi-soft cheeses were produced by the

coagulation of raw ewe milk, added with salt, using vegetable

rennet (cardoon flower) during a 40- to 60-day ripening period.

Cheese ripening is carried out on wooden shelve, in maturation

rooms at low temperatures (6–14 -C) and at a relatively high

humidity (80–95%). The cheeses are turned upside down, at

each day and, normally, after 1 week, a viscous smear appears

on the surface of the cheese. From this time to the end of the

ripening period, the cheese are washed and tied up with clean

cotton straps, almost daily.

In addition to the cheese samples, this survey also included

the collection of over 300 samples from cheesemaking envi-

ronments (walls, drains, equipment and utensils) and milking

environments (filters, bulks, nipples), after sanitizing opera-

tions, from bulk milk, milk from individual animals, animal

feed and faeces, and water (portions of 5000 ml). Samples from

walls, drains, equipment and utensils were collected either by

swabbing (Difco cotton swabs, Difco Laboratoires, Detroit,

USA), or by contact surface Palcam plates (Merck, Darmstadt,

Table 1

The source of L. monocytogenes isolates

Number of

isolates

Date of

isolation

Origin Farmhouse

1 24-07-1997 Blood –

1 09-09-1997 CSF –

1 20-10-1997 CSF –

1 08-04-1998 CSF –

1 13-01-1999 CSF –

1 16-07-1999 CSF –

1 20-09-1999 Blood –

1 31-03-2000 Blood –

1 15-06-2000 CSF –

1 13-04-2000 Blood –

1 16-10-2000 Blood –

1 24-05-2001 Blood –

1 07-11-2001 Blood –

1 14-03-2002 Blood –

1 25-02-2003 CSF –

2 26-06-2002 Plug of the refrigeration tank A

2 26-06-2002 Washing brush A

2 23-10-2002 Bulk milk A

2 23-10-2002 Milking device A

1 23-10-2002 Dairy equipment A

4 26-11-2002 Cheese (1) A

2 19-02-2003 Cheese (2) A

2 19-02-2003 Cheese (3) A

4 05-03-2003 Bulk milk A

2 26-03-2003 Bulk milk A

2 02-04-2003 Bulk milk A

3 26-03-2002 Cheese (1) B

3 02-04-2002 Cheese (1) B

3 30-04-2002 Cheese (1) B

2 04-06-2002 Cheese (2) B

2 28-05-2002 Cheese (3) B

1 09-07-2002 Cheese (4) B

1 09-04-2002 Cheese (1) C

2 16-04-2002 Cheese (1) C

4 28-05-2002 Cheese (2) C

6 04-06-2002 Cheese (2) C

3 18-06-2002 Cheese (2) C

1 26-11-2002 Cheese (3) C

1 04-12-2002 Cheese (4) C

3 21-01-2003 Cheese (5) C

2 16-04-2002 Cheese (1) D

3 28-05-2002 Cheese (2) D

1 04-06-2002 Cheese (2) D

4 09-07-2002 Cheese (2) D

1 26-06-2002 Wooden shelf D

2 22-10-2002 Milking device D

2 23-10-2002 Milking device D

3 04-06-2002 Cheese (1) E

2 23-10-2002 Milking device E

4 26-11-2002 Cheese (2) E

4 26-03-2003 Cheese (3) E

2 16-06-2003 Cheese (3) E

1 28-05-2002 Cheese F

3 15-01-2003 Cheese G

CSF=cerebrospinal fluid. For each farmhouse, the parenthesized numbers

mean different batches of cheese.

P. Leite et al. / International Journal of Food Microbiology 106 (2006) 111–121 113

Germany), laboratory made. Contact plates were sealed and

transported to the laboratory with the rest of the samples, under

refrigerated conditions (4–8 -C). All the samples were analysed

in less than 24 h for the presence of L. monocytogenes. Water

samples were filtered through sterile cellulose membranes of

0.22 Am of pore diameter (Millipore, Bedford, USA). Mem-

branes, swabs and all the other samples were immediately

submitted to the enrichment procedures. Plates were unsealed

and incubated at 37 -C for no less than 48 h.

2.3. Collection of human isolates

Between July 1997 and February 2003, 15 human isolates,

from cases of listeriosis, were collected in a public hospital

from the central part of Portugal. The strains were isolated from

blood, or from cerebrospinal fluid (CSF), mostly from elderly

and immunocompromised patients, some of them with associ-

ated cases of cirrhosis. The clinical history of the patients was

not available, as listeriosis is not reportable in Portugal.

2.4. Listeria monocytogenes detection and identification

This study used 114 L. monocytogenes strains, 5 of which are

reference strains obtained from CECT (Collecion Espanola de

Cultivos Tipo) and CIP (Collection de l’Institut Pasteur):

CECT4031T (serovar 1a), CECT936 (serovar 1/2b), CECT

911 (serovar 1/2c), CECT4032 (serovar 4b) and CIP104794

(=NCTC7973) (serovar 1/2a). Table 1 shows the source of the

109 L. monocytogenes isolates: the 94 non-clinical isolates

analysed here, selected according to different farms, collection

time, isolation procedures and origin. Eight were isolated from

the milking environment (milk filters), six from the dairy

environment (milk filters, milk refrigeration tank, cheese

washing brushes, and wooden shelve), ten from milk and

seventy were isolated from cheese. Detection of L. monocyto-

genes was generally performed on cheese and milk, according

to the vertical standard IDF143A (1995) and on environmental

samples, according to the horizontal standard ISO 11290-1

(1998). From each sample, three to six colonies were recovered.

The 15 human isolates were collected from patient’s blood or

CSF as follows: blood cultures from patients were incubated in

the automated BacT/ALERT system (Biomerieux, Marcy

L’Etoile, France). Positive samples were subcultured on

Columbia CNA agar with 5% sheep’s blood, and on 5% sheep’s

blood agar (Biomerieux), and incubated for 24 h to 48 h.

Samples from CSF were cultured, after centrifugation, on 5%

sheep’s blood agar and on chocolate agar plates and in Brain

Heart Infusion broth (Biomerieux). Species identification of the

non-clinical Listeria clones was performed combining the use

of the chromogenic selective medium ALOA (Ottaviani and

Agosti, 1997) and horse blood agar (HBA) (AES Laboratoire,

Bruz, France) with a PCR based assay (Furrer et al., 1991).

Presumptive colonies on Palcam agar (Merck) and/or Oxford

agar medium (Oxoid, Hampshire, UK) were further isolated by

re-streaking on TSA-YE (Oxoid), submitted to Gram staining,

catalase test, picked on HBA and ALOA. Positive colonies,

both on blood agar and ALOA, were further confirmed by API-

Listeria (Biomerieux) and tested by PCR assay. The clinical

isolates were identified based on colony morphology, and by

using conventional biochemical tests (beta-haemolysis, Gram

staining, catalase test, motility test) together with a commercial

system (API-Coryne strip, Biomerieux).


2.5. Serotyping

Serotyping of the strains was performed by the standard

method of Seeliger and Hohne (1979).

2.6. RAPD analysis

RAPD primers UBC127 (ATCTGGCAGC) and UBC155

(CTGGCGGCTG) (Farber and Addison, 1994), and HLWL85

(ACAACTGCTC) (Wernars et al., 1996) were synthesized

from MWG-Biotech AG, Switzerland. Amplifications with

primers UBC127 and UBC 155 were carried out as previously

described by Farber and Addison (1994), and amplifications

with primer HLWL85 were, basically, according to Vogel et al.

(2001), except that MgCl2 concentration used was 4 mM, as

suggested by Munthali et al. (1992), as a mean to increase the

number and intensity of the RAPD bands. A total volume of 25

Al was used, from which 1 Al corresponded to the DNA

bacterial lysate obtained, by boiling the cells in the presence of

Triton X-100 (Sigma, Steinheim, Germany), as described in

Cabrita et al. (2004). With primers UBC 127 and UBC 155, the

PCR mixture contained 4.0 mM MgCl2 (Invitrogen, Life

Technologies, Barcelona, Spain), 0.2 mM of each dNTP

(Promega, Madison, USA), 1.0 AM of primer and 1.25 U of

Taq DNA polymerase (Invitrogen, Life Technologies, Barce-

lona, Spain). With primer HLWL85, the PCR mixture was

similar, except for the primer concentration that was 4 AM.

PCR was carried out in a thermocycler Stratagene 96

(Stratagene cloning systems, La Jolla, CA, USA) for 1 cycle

at 94 -C for 2 min followed by 34 cycles of 94 -C for 1 min, 35

-C for 1 min and 72 -C for 2.5 min, with a final extension of 5

min at 72 -C (with primers UBC 127 and UBC 155), and 1

cycle at 94 -C for 2 min followed by 45 cycles of 1 min at 95

-C, 2 min at 35 -C, and 1 min at 72 -C, and then 1 cycle at 72

-C for 10 min (with primer HLWL85). Amplification products

were visualized after electrophoresis in a 1.5% agarose gel by

staining with ethidium bromide. The gels were analysed by

using the Bio-Rad Gel Doc 2000i (Bio-Rad Laboratories,

Segrate, Milan, Italy).

2.7. PCR with specific primers for the gene actA

The reaction volume of 50 Al for PCR contained 1 Al of theDNA solution extracted by using the ‘‘High Pure PCR

Template Preparation Kit’’ (Roche, Mannheim, Germany).

The primers used were PR5 (TGA AGA GGT AAA TGC

TTC GGA CTT) and PR3 (CGC TTA TTT TCG GTA CCT

TTG GA) designed according to Moriishi et al. (1998). The

PCR mixture contained 2.0 mM MgCl2 (Invitrogen, Life

Technologies, Barcelona, Spain), 0.2 mM of each dNTP

(Promega, Madison, USA), 0.5 AM of each primer (Sigme-

Genosys, Cambridge, UK) and 2.5 U of Taq DNA polymerase

(Invitrogen, Life Technologies, Barcelona, Spain). PCR was

carried out, according to Inoue et al. (2001), in a thermocycler

Stratagene 96 for 1 cycle at 92 -C for 5 min, followed by 25

cycles (92 -C for 1 min, 45 -C for 1 min, and 74 -C for 2 min)

but with a final extension of 10 min at 72 -C. Amplified DNA

fragments were further resolved in a 1.5% 0.04 M Tris–acetate,

0.001 M EDTA agarose gel (Sigma, Steinheim, Germany) at

4.3 V/cm for 90 min. The gels were stained and analysed as

described above.

2.8. PFGE

The 109 L. monocytogenes isolates were characterized by

DNA macrorestriction patterns obtained after separate diges-

tion with AscI and ApaI, respectively, and separation of the

generated fragments by pulsed-field gel electrophoresis

(PFGE) with a previously described protocol (Graves and

Swaminathan, 2001).

2.9. Data analysis

Similarities between banding patterns were established with

NTSYS program (Rohlf, 1987) using unweighted pair-group

method with arithmetic averages (UPGMA) clustering, based

on the Dice correlation coefficient (Priest and Austin, 1993).

The index of discrimination of the isolates, expressed as a

percentage, was determined as described by Hunter and Gaston

(1988) and Dillon et al. (1993).

3. Results

3.1. Listeria monocytogenes in farmhouse cheese manufactures

Samples collected at different time periods, within two

consecutive cheesemaking seasons, from seven farmhouses (A

to G, in Table 1) showed the presence of this pathogenic

bacterium. In farmhouse A, L. monocytogenes was only

detected in the second cheesemaking period. Farmhouse B,

which showed the presence of the pathogen during the first

period, was able to eradicate L. monocytogenes during the

second season. One of the farmhouses (G) stopped activity and

farmhouses C, D, E and F were positives for the presence of L.

monocytogenes during both sampling periods. None of the

seven smallest farmhouses (flocks with less than 80 sheep) was

positive for L. monocytogenes. The prevalence of L. mono-

cytogenes in the surveyed farmhouses was about 25%.

3.2. Serotyping

Serotyping indicated the presence of four different serovars

among the isolates: 1/2a, 1/2b, 1/2c and 4b (Table 2). The 15

clinical isolates were either serovar 4b (86.7%) or serovar 1/2b

(13.3%). At the seven farmhouses with positive results for the

presence of L. monocytogenes, the serovars prevalence was 1/

2a (1.1%), 1/2b (17.0%), 1/2c (12.8%) and 4b (69.1%).

3.3. RAPD typing

Initial screening of six different primers (DAF4, HLWL85,

OMP-01, UBC127, UBC155 and UBC156) was performed

with 31 L. monocytogenes isolates and three reference strains

(serovars 1/2b, 4b and type strain) (results not shown). Three of

Table 2

Number of L. monocytogenes RAPD types, obtained with three different

primers, and percentages of actA gene types I and II, for different serovars

Serovar Number

of strains

Number of RAPD types actA gene type (%)

UBC127 UBC155 HLWL 85 All I II

1a 1 1 1 1 1 1* (100) 0 (0)

1/2a 2 2 1 1 2 2* (100) 0 (0)

1/2b 19 3 2 3 3 19* (100) 0 (0)

1/2c 13 2 1 2 2 13* (100) 0 (0)

4b 79 7 2 5 14 68 (86.1) 11* (13.9)

Total 114 14 4 9 21 103 (90.4) 11 (9.6)

The asterisk on the number of strains means that the reference strain is

included.


these primers (HLWL85, UBC127 and UBC155) displayed

more discernible and discriminatory profiles and for this

reason were selected to type L. monocytogenes isolates.

Reproducibility of RAPD assays was assessed by performing

at least two independent trials. Low intensity bands were not

consistently reproducible and were not taken into consider-

ation when the RAPD patterns were compared. No amplifi-

cation products were detected with the negative control (water

instead of cell lysate), confirming the absence of contamina-

tions. Fig. 1 shows the RAPD patterns of L. monocytogenes

strains obtained with primer UBC127. From the 114 strains

examined (109 isolates and 5 reference strains), 4, 9 and 14

different RAPD types were observed with primers UBC155,

HLWL85 and UBC127, respectively (Table 2). By the use of

the three primers, 21 types were generated (Tables 2 and 3). If

only the 109 isolates were considered, the result would be 18

RAPD types (Table 3). The number of RAPD bands,

Fig. 1. RAPD typing of L. monocytogenes strains, with primer UBC127: (1–5) refe

12) cheese and (10, 13, 14 and 15) cheese-related isolates from serovar 4b; (18–20) h

cheese isolate from serovar 1/2a; (16) cheese isolate from serovar 1/2c; (M) molecula

produced for a given primer, ranged from 2 to 10, with

molecular sizes from 0.3 to 5.2 kb. With the three primers,

the total number of types obtained with all the strains tested

was inferior to the sum of the number of types per serovar

(Table 2). This fact was due to the sharing of banding profiles

among strains of different serovars namely, with the three

primers, the type strain (CECT4031T, serovar 1a) and the

reference strain for serovar 1/2a (CIP104794) displayed

identical profiles. In addition, with primers UBC155 and

HLWL85, respectively, serovars 4b and 1/2b shared identical

patterns, and with primer HLWL85 the serovar 1/2c reference

strain showed the same RAPD profiles as the type strain and

the serovar 1/2a reference strain (data not shown). With the

exception of primer UBC155, the RAPD primers were able

do differentiate isolates within the same serovar (Table 2).

Except for the type strain and for serovar 1/2a reference

strain, the overlapping among serovars observed with the

individual use of the three primers was overpassed by the

combined use of the three primers.

3.4. actA gene typing

PCR amplification of the proline-rich region (PRR) with

primers PR5 and PR3 differentiates two alleles, characterized

by the presence of one proline-rich unit (LU) or two LUs.

According to Inoue et al. (2001), L. monocytogenes strains

with one or two LUs are actA type II (518 bp amplification

product) or type I (623 bp amplification product), respectively

(Fig. 2). The percentage of isolates belonging to each gene type

is indicated in Table 2. One hundred and three (90.4%) of one

rence strains for serovars 1a, 4b, 1/2c, 1/2b and 1/2a, respectively; (6, 7, 11 and

uman isolates from serovar 4b; (8 and 17) cheese isolates from serovar 1/2b; (9)

r standard ‘‘1 kb DNA ladder’’ (Invitrogen) and (C) negative control with water.

Table 3

Molecular types obtained from 109 isolates, from humans and from cheese and

cheese-related isolates, and five reference strains of L. monocytogenes, from

different serovars

Number of isolates Molecular types Serovar

PFGE RAPD actA

1 nd A I 1aa

1 nd B II 4ba

1 nd C I 1/2ca

1 nd D I 1/2ba

1 nd A I 1/2aa

48 6 E I 4b

1 11 E I 4b

16 3 D I 1/2b

1 1 F I 1/2a

1 10 G I 4b

3 9 G I 4b

8 7 E I 4b

2 8 H II 4b

2 12 I II 4b

12 2 J I 1/2c

1 20 K II 4b

1 21 L II 4b

1 22 M I 4b

1 23 N I 4b

1 5 O I 1/2b

1 24 G I 4b

1 4 P I 1/2b

1 17 Q II 4b

1 19 K II 4b

1 14 Q II 4b

1 13 R I 4b

1 15 S I 4b

1 16 T I 4b

1 18 U II 4b

1 25 R I 4b

nd=not determined. Bold=RAPD types of the isolates that were further

discriminated by PFGE analysis.a Serovars signed with an asterisk correspond to the reference strains.


hundred and fourteen isolates were identified as actA gene type

I, and eleven (9.6%) isolates as actA gene type II (Table 2).

3.5. PFGE typing

One hundred and nine L. monocytogenes isolates, collected

from cheeses and cheese-related environments and from

human cases of listeriosis, were typed by PFGE by using

the enzymes ApaI and AscI. Fig. 3 displays PFGE profiles of

L. monocytogenes DNAs after macrorestriction with the

enzyme ApaI. From the 109 strains examined, 25 and 23

genotypes were observed with the enzyme ApaI and AscI,

respectively. By the use of the two enzymes, 25 genotypes

(1–25) were generated (Table 3 and Fig. 4). The number of

bands, produced with ApaI and with AscI, ranged from 12 to

17, with molecular sizes from 40 to 439 kb, and 7 to 12, with

13–700 kb in length, respectively. The relationships among

L. monocytogenes genotypes based on their PFGE profiles are

shown in the dendrogram displayed in Fig. 4, and are

supported by a high cophenetic correlation coefficient

(q =0.92). The 25 L. monocytogenes genotypes studied could

be divided into two major clusters, at about 0.20 similarity

level. The major cluster was composed of strains from

serovars 1/2b and 4b, and the other cluster was represented

by the cheese and cheese-related isolates from serovars 1/2a

and 1/2c. Serovar 4b displayed 7 different types among 65

isolates, from a total of 94 cheese and cheese-related isolates

(Table 4 and Fig. 4), and 13 different types among 13

isolates, from a total of 15 human isolates (Fig. 4), showing a

great genetic diversity even though, this was the serovar with

the highest number of isolates (71.6%), both cheese and

cheese-related, and humans. Serovar 1/2b was the second

more frequent serotype among cheese and cheese-related

isolates (16/94) and the other serovar displayed by the clinical

isolates (Table 4 and Fig. 4). Genotype 3 was produced by

serovar 1/2b isolates from farmhouses B and G (Table 4 and

Fig. 4), and isolates from farmhouses A, C and D, shared

serovar 4b type 6 (Table 4 and Fig. 4). No sharing of

genotypes was observed among human isolates, and also

there was no common genotype between isolates from human

cases and from farmhouses (Fig. 4). The discriminatory power

of PFGE with the enzymes ApaI and AscI was, in both cases,

0.77. By using the two enzymes, the Simpson’s index of

diversity (SID) for the PFGE did not change, although the use

of the enzyme ApaI produced two more types (25) than the

enzyme AscI (23). The data displayed in Table 4 shows that

genotypes 6, 3 and 2, includes 48, 16 and 12 isolates,

respectively. This fact accounts for the relatively low SID

value, which is sensitive not only to the number of groups,

defined by the typing scheme, but also to the size of the

largest group. The assignment of a large number of isolates to

a few groups may reflect the clonal nature of these isolates

and their resident nature within farmhouse’s environments.

3.6. Combined results

In this study, 109 L. monocytogenes isolates and 5

reference strains were characterized. The isolates were from

raw ewe’s milk cheese, cheese-related environments, and

humans. RAPD analysis proved to be discriminative (18

combined RAPD types from the 109 isolates) (Table 3)

allowing strain differentiation within a serovar, especially

when using the three different primers. Nevertheless, PFGE

proved its superior discriminatory power, in differentiating the

same number of isolates (25 combined PFGE types) (Table

3). The analysis of the Table 3 shows that L. monocytogenes

strains from the five RAPD types, E, G, K, Q and R, were

further differentiated by PFGE in 12 PFGE types (6, 7 and

11; 9, 10 and 24; 19 and 20; 14 and 17; 13 and 25,

respectively). From the three RAPD primers, UBC127 was

the most discriminatory, rendering 14 types (Table 2), but the

combined used of the three primers was essential to raise the

discriminatory power of the RAPD analysis. With PFGE, the

use of the enzyme ApaI produced 25 types, and with the

enzyme AscI, only 23 types were displayed. Clustering of the

strains based on their PFGE profiles (Fig. 4), showed the

assembly of strains from serovar 1/2b and 4b into one major

group (lineage I). It was within this cluster that were gathered

the eleven isolates belonging to actA gene type II (Fig. 4).

Fig. 2. actA gene typing of L. monocytogenes strains. The primers used were PR5 and PR3 which flank the region of the gene that encodes the two LUs (large units):

(1–5) reference strains for serovars 1a, 4b, 1/2c, 1/2b and 1/2a, respectively; (6, 7, 11 and 12) cheese and (10, 13, 14 and 15) cheese-related isolates from serovar 4b;

(18–21, 23 and 25–32) human isolates from serovar 4b; (8 and 17) cheese isolates from serovar 1/2b; (22 and 24) human isolates from serovar 1/2b; (9) cheese

isolate from serovar 1/2a; (16) cheese isolate from serovar 1/2c; (M1 and M2) molecular standards ‘‘1 kb DNA ladder’’ (Invitrogen) and ‘‘DNA molecular weight

marker XIV (100–1500 bp)’’ (Roche), respectively and (C) negative control with water.


They were all serovar 4b, and represented 13.9% of the 79

isolates from this serovar (Table 2). From these eleven

isolates, 6, out of 15, were of human origin (Figs. 2 and

Fig. 3. PFGE typing of L. monocytogenes strains, with the enzyme ApaI: (1 and

respectively; (2) human isolates from serovar 1/2b (PFGE type 5); (7, 8 and 13–15)

serovar 1/2c (PFGE type 2) and (M) molecular standard is L. monocytogenes strain

4), 4, out of 94 cheese-related isolates, were collected from

the milking device of producer D (Tables 1 and 4 and Fig. 4)

and 1 was the reference strain for serovar 4b (CECT4032)

3–6) human isolates from serovar 4b (PFGE types 24, 23, 22, 21 and 20,

bulk milk isolates from serovar 4b (PFGE type 6); (9–12) cheese isolates from

number CLIP 77873 (CLIP: Listeria Collection of the Pasteur Institute).

Coefficient

0.15 0.36 0.57 0.78 0.99

1

2

3

6

7

11

9

8

12

23

20

21

22

10

5

24

4

13

15

16

25

18

14

19

17

PFG

E T

YPE

1/2a

SER

OV

AR

1/2b

4b

4b

4b

4b

4b

4b

4b*

4b*

4b*

4b*

4b

1/2b*

4b*

1/2b*

4b*

4b*

4b*

4b*

4b*

4b*

4b*

4b*

1/2c

FAR

MH

OU

SE

B1

B1,G1

A3,C1,D1

A3

D1

E1

D2

D2

F1

E3

07-02

03-02/01-03

ISO

LA

TIO

ND

AT

E(m

m-y

y)

04-02/04-03

11-02

06-02

06-02

10-02

10-02

10-00

03-00

06-00

04-00

05-02

03-02

05-01

11-01

07-97

10-97

04-98

02-03

07-99

09-97

09-99

01-99

11-02/06-03

actA

TY

PE

I

I

I

I

I

I

II

II

I

II

II

I

I

I

I

I

I

I

I

I

II

II

II

II

I

Fig. 4. Dendrogram (UPGMA clustering based on Dice correlation coefficient) of Listeria monocytogenes ApaI and AscI PFGE profiles (163 bands) for 25

genotypes, corresponding to 109 isolates from cheese and related environments and from humans. The 15 types, which correspond to the 15 human isolates, are

signed with an asterisk on serovar discrimination. In each farmhouse, the L. monocytogenes genotypes were isolated from the: 1–dairy environment; 2–milking

environment or 3–dairy environment and milking environment.


(Tables 2 and 3 and Fig. 2). All the strains from serovars 1a,

1/2a, 1/2b and 1/2c were actA gene type I (Table 2).

4. Discussion

We have investigated Portuguese farmhouse cheese manu-

factures, from the central part of Portugal, for the presence of L.

Table 4

Number of L. monocytogenes combined PFGE types obtained, from cheese and

cheese-related isolates, for different serovars

Serovar Genotype Number of isolates per farmhouse

A B C D E F G All

1/2a 1 0 1 0 0 0 0 0 1

1/2b 3 0 13 0 0 0 0 3 16

1/2c 2 0 0 0 0 12 0 0 12

4b 6 17 0 21 10 0 0 0 48

7 8 0 0 0 0 0 0 8

8 0 0 0 2 0 0 0 2

9 0 0 0 0 3 0 0 3

10 0 0 0 0 0 1 0 1

11 0 0 0 1 0 0 0 1

12 0 0 0 2 0 0 0 2

Total 25 14 21 15 15 1 3 94

monocytogenes and looked for causal associations between the

consumption of the cheese produced there and cases of

listeriosis from the same geographical region. We characterized

109 isolates from both origins, collected in overlapping dates,

and found a prevalence of serovar 4b, both within cheese and

cheese-related isolates (69.1%), and among human isolates

(86.7%). The serovars distribution for the cheese isolates was

not expected, because among strains isolated from food

products serogroup 1/2 has been reported to dominate (Farber

and Peterkin, 1991; Loncarevic et al., 1995). Guerra et al.

(2001), in a study of the incidence of Listeria species in

different ready-to-eat and unprocessed foods produced in

Portugal, found a prevalence of isolates from serovars 1/2a

and 1/2b (78%). But in a survey made in 1995–1996, in the

same geographical region analysed here, Pintado et al. (2005)

also detected a prevalence of serovar 4b (83%), among isolates

from a soft cheese of a similar type. These results may suggest

the endemic nature of the L. monocytogenes isolates associated

with the particular characteristics of this type of ewe’s cheese.

The association of serovars 1/2b and 4b with listeriosis, in

humans, has been well documented (McLauchlin, 1990;

Schuchat et al., 1991) and the serotyping of these Portuguese

human isolates is in accordance with these previous reports.


The high prevalence of these same serovars in ewe’s cheese

triggered the use of more discriminatory typing methods in

order to investigate the relationships among these L. mono-

cytogenes cheese and clinical isolates.

The combined use of RAPD and PFGE analysis have

previously proved to be a valuable tool to trace the

dissemination of L. monocytogenes in food plants (Destro et

al., 1996; Giovannacci et al., 1999; Chasseignaux et al., 2001).

In this study, the PFGE typing showed greater discriminatory

power among L. monocytogenes isolates (25 types) than RAPD

typing (18 types) (Table 3). But, as already reported by

Gudmundsdottir et al. (2004) in PFGE typing of L. mono-

cytogenes isolates from serovars 1/2a, 1/2b and 4b, the use of

the enzyme AscI did not add further diversity to the data

obtained by using ApaI. The RAPD protocol, especially when

using bacterial cell lysates, is very fast and relatively less

expensive when compared at cost price with PFGE analysis.

Nevertheless, and depending on the primer and on the strain,

the faintness of some bands may render some profiles of

difficult interpretation. For this reason, but mainly because

RAPD revealed less discriminatory power on these strains,

only the PFGE profiles were considered to study the genetic

relationships among L. monocytogenes isolates. In previous

work, Cabrita et al. (2004) by using, in a total of three RAPD

primers, two of the primers used in this study (UBC127 and

UBC155) achieved a good discriminatory power, although

there was no differentiation within serovar 4b. In that study, the

strains from this serovar were only 18% of the total strains

analysed, and in the present study the strains from serovar 4b

represent 69% of the total analysed strains. This fact may

reflect the lack of accuracy of the primers used in strain

differentiation within serovar 4b. The clustering of L. mono-

cytogenes serovars into two groups, based on their PFGE

profiles (Fig. 4), was in accordance with previous assignment

of L. monocytogenes serovars into three lineages. The major

cluster corresponds to lineage I, with serovars 1/2b and 4b, and

in the other cluster, at 0.20 similarity, are gathered the isolates

from serovar 1/2a and 1/2c, from lineage II.

Typing of the actA gene in 114 strains yielded 2 profiles

(type I or type II, according to Inoue et al., 2001) (Figs. 2 and 4

and Table 2). These two types correspond to Wiedmann et al.

(1997) allele 4 and allele 3, respectively. Jeffers et al. (2001), in

a study with clinical L. monocytogenes isolates, reported a

higher frequency of actA allele 4 (actA gene type I) within

lineages II (92%) and III (100%) than within lineage I (39.6%).

Previously, Wiedmann et al. (1997) had similar results, with

higher percentages of strains belonging to actA allele 4 (42/47)

in lineage II, than in lineage I (12/70). Inoue et al. (2001), in a

study that included L. monocytogenes isolates from humans

and from the intestinal contents of cows and beef, detected

actA gene type II more frequently in serovar 4b (46.9%), from

lineage I, than in serovar 1/2b (7.7%), from the same lineage,

or among serovars 1/2a (22.2%) and 1/2c (0%), from lineage II.

In the present study from 25 L. monocytogenes genotypes (109

isolates), 10 types from cheese and cheese-related environ-

ments and 15 types from humans, we detected actA gene type

II in eight genotypes (six types from humans strains and two

types from non-human isolates) (Fig. 4), corresponding to

eleven isolates from serovar 4b. The types belonging to

serovars 1/2a, 1/2b and 1/2c were all actA gene type I (Table

2 and Fig. 4). Our results are more in agreement with the results

of Inoue et al. (2001). The fact that the majority of our isolates

were from serovar 4b could explain the apparent discordance

with the results of Wiedmann et al. (1997) and Jeffers et al.

(2001).

Gudmundsdottir et al. (2004), on a study encompassing 10

Icelandic sheep farms, found a major percentage of isolates

from serovar 1/2a, some of them with associated cases of

listeriosis in sheep, and suggest that this could be due to the

predominance of this serovar in silage. In more than one farm,

they found nine L. monocytogenes genotypes. In the present

study, the distribution of genotypes, in farmhouses cheese, was

relatively homogeneous. Whenever, from the same sample,

more than one isolate was obtained, from different isolation

media and procedures, only one genotype was recovered.

Farmhouses A, C, D and E were investigated and found

positive for L. monocytogenes detection during 1 year (that

encompassed two cheesemaking seasons). Farmhouse C

harboured only one genotype (6, serovar 4b), also shared by

farmhouses A and D in the surveyed period (Fig. 4), even with

a strict effort put on cleaning and disinfection procedures,

namely on cheese production environment. In general, in the

seven farmhouses, the equipment and the environment were

not very contaminated because we only were able to collect L.

monocytogenes environmental isolates from farmhouses A, D

and E (Table 1). In farmhouse A, two genotypes were identified

(6 and 7, serovar 4b), but the 89% similarity level shared by

these types suggested the clonal origin of the corresponding

isolates (Fig. 4). PFGE types 6 and 7 were not discriminated by

RAPD analysis, and corresponded to RAPD type E (Table 3).

Farmhouse D was the one that presented the higher number of

genetic types (6, 8, 11 and 12, serovar 4b) (Table 3 and Fig. 4).

However, types 8 and 12 harboured closely related strains (Fig.

4), and type 11 was only distinguished from type 6 by PFGE

(Table 3). In farmhouse E, two genotypes were found, 2 and 9,

from serovars 1/2c and 4b, respectively (Fig. 4). In farmhouses

A and E, the existence of the same genotypes, both in milking

and in cheese making environments (Fig. 4), suggests that L.

monocytogenes enter into the cheese manufacture carried by

the milk. The presence of these bacteria in the milk may result

from direct excretion from the udder or as a result of

environmental contamination. In flocks from farmhouses A

and D, we have investigated the milk from suspected animals

(somatic cell counts—SCC>500,000/ml) and L. monocyto-

genes was not found (Moura et al., 2004). This may suggest

that the more frequent cause of L. monocytogenes contamina-

tion in these farmhouse ewe cheese manufactures is environ-

mental contamination of the milk during milking. Sheep, as

almost any animal species, can carry L. monocytogenes even

asymptomatically (Husu, 1990; Iida et al., 1998; Gudmunds-

dottir et al., 2004). Although in dairy cattle the involvement of

L. monocytogenes in product contamination has often been

associated with feeding with poorly fermented silage (Vazquez-

Boland et al., 1992; Wiedmann et al., 1994), this is not the case


here. The fact that some genotypes are common to different

farmhouses, which are apart from each others by at least 30 km,

and do not share flocks or cheese manufactures, suggests that

these genotypes are widely spread. It would appear that some

Listeria clones became endemic as a result of an adaptation to

specific ecological niches. This fact reinforces previously

observations in other food industries concerning the persistence

of particular clonal types (Giovannacci et al., 1999; Chas-

seignaux et al., 2001). Three of the isolates analysed here, from

serovars 1/2b and 4b, showed good growth behaviour at near

alkaline pH (Manha et al., 2003). This result may reflect the

exposure of L. monocytogenes cells to detergents and disin-

fectants, usually alkaline, used to sanitize facilities and

equipment. These alkaline-stressed adapted cells may become

difficult to eradicate (Taormina and Beuchat, 2001). In face of

our results and of previous reports for the persistence of L.

monocytogenes in dairy environments (Unnerstad et al., 1996)

we could expect that these persistent clones may be residents in

the farmhouses even before the beginning of our survey.

When analysing the genetic relationships between clinical

and food related isolates we found no common genotypes. The

15 human strains analysed in this study correspond to all the L.

monocytogenes collected in this hospital from September 1997

to February 2003. Because in Portugal there is no surveillance

for listeriosis, we could only hypothesize that the farmhouses

analysed here were not involved in these cases of listeriosis.

The dendrogram displayed in Fig. 4 shows that among clinical

strains, there were isolates with the same clonal origin. This is

the case of types 14, 17 and 19 (actA gene type II), which

included isolates from 1997 to 1999, clustered at 93%

similarity, types 20 and 21 (actA gene type II) both from

2000, with 3 months apart, clustered at 90% similarity, and

type 15 and 16 (actA gene type I), with isolation dates with 6

months apart, grouped at 96% similarity. It is possible that

these isolates will be resident strains in particular food plants.

Nevertheless, the prevalence of serovar 4b, and to a lesser

extent of serovar 1/2b, in ewe cheese is of particular concern.

During the last years, a great improvement was made, in

Portugal, on cheese manufactures facilities and equipments,

along with a substantial improvement in employing good

manufacturing practices (GMPs). Regrettably, the same im-

provement was not achieved in herd management, particularly

in udder health preventive management. Most of the farm-

houses do not dispose of a parlour, and for this reason milking

is performed at the housing barn, with no running water

available, and in deficient illumination conditions. On these

circumstances, it is very difficult to accomplish hygiene

standards. In spite of these restrictions, 75% of the screened

farmhouses were L. monocytogenes-free. Strains from serovars

1/2b and 4b belong to lineage I, which comprises the more

virulent L. monocytogenes strains, although in previous work

we have presented results that support the existence of

heterogeneity in virulence potential within serovar 1/2b

(Cabrita et al., 2004). The virulent potential of these isolates

will be further investigated, as it is not known whether their

resident condition may, some how, interfere with their

pathogenic potential. In the manufacture of cheese from raw

milk as well as from pasteurized milk, it is necessary to analyse

the entire process and to identify the critical control points. To

rely only on pasteurisation to solve L. monocytogenes

contaminations is likely to limit efforts to improve total

hygiene in food production. Producer’s education and technical

support, because some of these cheeses are DOP produce

(denomination of protected origin), rigorous inspection, clear

labelling including use by/best before dates, together with

informed consumer choice will be the lead to strength

consumer confidence in the safety of these high standard

gourmet cheese.

Acknowledgments

This study was based on research conducted under Project

292, PO AGRO 8.1-10 Concurso, supported by Instituto

Nacional de Investigacao Agraria e das Pescas. Pedro Leite

and Rui Rodrigues were recipients of grants from PRODEP III.

The authors would like to thank Ana Carla Silva for

technical assistance and Joao Madanelo for stimulating

discussions concerning ewe’s milk and ewe’s cheese produc-

tion policies and technologies.

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