ARTICLE

Distribution and characterization of Listeria monocytogenesclinical isolates in Portugal, 1994–2007

G. Almeida & A. Morvan & R. Magalhães & I. Santos &

T. Hogg & A. Leclercq & P. Teixeira & Research Team

Received: 19 January 2010 /Accepted: 29 May 2010# Springer-Verlag 2010

Abstract In recent years, the number of cases of listeriosishas increased worldwide. Ninety-five isolates of Listeriamonocytogenes recovered from Portuguese human cases oflisteriosis have been characterized by biotyping (cadmiumand arsenic sensitivity), polymerase chain reaction (PCR)grouping, and by pulsed-field gel electrophoresis (PFGE)applying the enzymes AscI and ApaI. Isolates wereclassified into one of three PCR groups; IVb (71.6%), IIb(17.9%), and IIa (10.5%). Four biotypes were differentiated:sensitive to arsenic/cadmium (48.4%), arsenic-sensitiveand cadmium-resistant (25.3%), resistant to arsenic andsensitive to cadmium (18.9%), and resistant to bothheavy metals (7.4%). Combined analyses of AscI andApaI patterns yielded a total of 58 PFGE types with fivesets (G, Jb, KKa, Me, and U) of Portuguese strains, eachof which were indistinguishable by PFGE typing. In thepresent study, it was demonstrated that there are recurrentpulsotypes and that some were the same pulsotypes linkedto outbreaks in France. In addition, there are somepulsotypes spread throughout the country, while othersonly appear in a restricted region. This study allowed theassembly of a first large pulsotype database of Portugueseclinical strains.

Introduction

Listeria monocytogenes is recognized as a major, opportu-nistic, foodborne pathogen in humans. The condition whichthis species causes, listeriosis, occurs worldwide bothsporadically and epidemically, and has a case–fatality ratebetween 20 and 30%. L. monocytogenes has the ability tocross the intestinal, blood–brain, and fetoplacental barriers,and, thus, septicemia, central nervous system (CNS)infections, miscarriages, and stillbirths are risks to immu-nodeficient or pregnant individuals [1].

In Portugal, listeriosis is not a notifiable disease and has,thus, been underestimated. Nevertheless, Almeida et al. in2006 [2], in a retrospective study, reported an estimatedincidence of at least 1.4 cases per million inhabitants in2003. The implementation of an active surveillanceprogram in the Netherlands led to a 43% increase in thereported incidence of listeriosis in the first year of operation[3]. This might give some indication of the current degreeof underestimation in Portugal. Any such program wouldcertainly omit reporting some sporadic cases and spontane-ous miscarriages, as these are often not investigated. Since2005, several European countries reported an increase oflisteriosis and several foodborne outbreaks have beenreported [4]. Some examples are the outbreaks whichoccurred in Switzerland associated with Tome cheese [5],in the United Kingdom associated with sandwich consump-tion [6], in the Czech Republic associated with soft cheese[7], and a recent multinational outbreak due to theconsumption of “Quargel” cheese in Austria and Germany[8].

The characterization of L. monocytogenes strains can bebased on its molecular subtyping by polymerase chainreaction (PCR) grouping or geno-serotyping [9] and bymacrorestriction pulsotype analysis of its DNA using the

G. Almeida : R. Magalhães : I. Santos : T. Hogg : P. Teixeira (*)Centro de Biotecnologia e Química Fina, Escola Superiorde Biotecnologia, Universidade Católica Portuguesa,Rua Dr António Bernardino de Almeida,4200-072 Porto, Portugale-mail: pcteixeira@esb.ucp.pt

A. Morvan :A. LeclercqLaboratoire des Listeria, Centre National de Référence desListeria et Centre Collaborateur de l’Organisation Mondiale de laSanté pour la Listériose d’Origine Alimentaire, Institut Pasteur,25, Rue du Docteur-Roux,75724 Paris cedex 15, France

Eur J Clin Microbiol Infect DisDOI 10.1007/s10096-010-0988-x

PulseNet protocol [10]. The use of subtyping methods todifferentiate strains (or subtypes) of L. monocytogenes hasimportant epidemiological applications: rapid, precise, andefficient foodborne listeriosis surveillance can minimizeoutbreaks and track sources of L. monocytogenes contam-ination throughout the food system. Moreover, molecularsubtyping in listeriosis surveillance would allow thedetection of clusters of cases that could be erroneouslyconsidered as sporadic cases.

The association between a particular pulsotype andspecific virulence traits is another possibility offered bymolecular subtyping [11].

The study presented here was initiated in 2003, with aview to support the establishment of an integrated, foodchain surveillance system in Portugal. Ninety-five clinicalPortuguese isolates recovered from apparently sporadiccases of listeriosis were collected, identified, biotyped bycadmium and arsenic sensitivity, geno-serotyped, andtyped by DNA macrorestriction pulsed-field gelelectrophoresis (PFGE). The aim was to obtainepidemiological data on cases of listeriosis in the countryand to construct a molecular type database of clinicalstrains of L. monocytogenes.

Materials and methods

Collection of L. monocytogenes strains and related datato listeriosis episodes

A total of 95 L. monocytogenes strains isolated between1994 and 2007 were collected from major Portuguesehospitals. The case definition of listeriosis employed wasthat contained in Commission Decision 2002/253/CE [12].Thus, on isolation of L. monocytogenes from a hospitalizedpatient with a clinical compatible illness, each strain wasconsidered as a single case of listeriosis. A case wasdefined as maternal/neonatal (MN) in the followingsituations: infected pregnant woman, miscarriage, stillbirth,or newborn less than one month of age. Only one strain waskept when the pathogen was isolated from both thepregnant woman and her newborn child. If a case did notapply to any of these, it was considered as non-maternal/neonatal (non-MN). Information regarding gender and ageof the patient, the tissue or fluid origin where the bacteriawas isolated, and the year of isolation was reported whenavailable.

Confirmed isolates of L. monocytogenes were stored inTryptic Soy Broth with 30% (v/v) glycerol at −80° Cin the culture collection of the Escola Superior deBiotecnologia.

Biotyping by arsenic and cadmium sensitivity

The characterization of their differential resistance toarsenic and cadmium was performed in Isosensitest agarplates (ISA, Oxoid, Hampshire, UK) containing 500 µg/mlsodium arsenite (Merck, Darmstadt, Germany) or 75 µg/mlcadmium chloride (Merck) [13, 14].

Geno-serotyping or PCR grouping

Geno-serotyping was determined by PCR grouping with amultiplex PCR as described byDoumith et al. [9] using primerstargeting fragments of genes lmo0737, ORF2819, ORF2110, lmo1118, and prs (MWG-Biotech, Muenchenstein,Switzerland). PCR was performed in an Eppendorfthermocycler (Eppendorf, Hamburg, Germany) and PCRproducts were resolved on a 2% agarose gel containing0.5 µg/ml of ethidium bromide (Eurobio, Courtaboeuf,France) and visualized and photographed under a UVtransilluminator (Bio-Rad Gel Doc 2000™ imagingsystem, Bio-Rad Laboratories, Milan, Italy).

Molecular characterization by PFGE

The DNA macrorestriction was performed at the CentreNational de Référence des Listeria, Institut Pasteur (Paris,France) with the restriction enzymes AscI (New EnglandBioLabs, Massachusetts, Ipswich, USA) and ApaI (MBIFermentas, Burlington, Canada), as previously described byGraves and Swaminathan [10].

The pulsotypes obtained were scanned and the comput-erized data were analyzed using BioNumerics softwareversion 5.1 (Applied Maths, Kortrijk, Belgium). Bandsautomatically assigned by the computer were checkedvisually and corrected manually when necessary. A positiontolerance of 1.5 was selected for each PFGE. Clusteranalysis of the individual or combined PFGE pulsotypeswas done by the unweighted pair group method withaverage linkages (UPGMA), using the Dice coefficient toanalyze the similarities of the banding pulsotypes. Thediscriminatory power was determined by calculating thediscrimination index (D) based on Simpson’s index ofdiversity, as described by Hunter and Gaston [15].

Two PFGE patterns were considered to be indistinguish-able when their similarity was higher than 98% [16] and acapital letter was ascribed to AscI pulsotype; when strainswere similar at more than 98% by AscI but not similar atmore than 98% by ApaI, they were denoted, in addition tothe capital letters, by lowercase letters.

PFGE pulsotypes of indistinguishable Portuguese strainswere compared with PFGE pulsotypes of the French PFGEdatabase of the Centre National de Référence des Listeria

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using the BioNumerics software package as previouslydescribed in this section.

Statistical analysis

The statistical package used for the analyses was Statav10.0 (StataCorp LP, College Station, TX, USA). Thecontingency table analysis was based on the Chi-squaredistribution (Pearson’s Chi-square test).

Results

Data collected from human episodes

A total of 95 isolates from cases of listeriosis werecollected from major Portuguese hospitals during thestudy period. From the information available on 81 cases(Table 1), 85.2 and 14.8% corresponded to non-MNinfections and MN infections, respectively. For the 69confirmed non-MN cases, strains were isolated from blood(58.0%), from cerebrospinal fluid (34.8%), and from otherspecimens (7.2%). The clinical symptoms were notrecorded for the majority of cases. The gender ratio (M/F) of confirmed non-MN cases was 2.3. The mean age ofnon-MN cases with documented age was 60 years, with 38cases (55.9%) being equal or up to 60 years. Thegeographical distribution of listeriosis cases was difficultto define from our collected data in the absence of data onthe residence of patients.

Biotyping results by arsenic and cadmium sensitivity

Four groupings of sensitivity to heavy metals weredifferentiated among the 95 isolates (Table 1), the D-valuefor this biotyping method being 0.667. The predominantgroup—sensitive to both arsenic (As) and cadmium (Cd)—contains 46 isolates (48.4%). Twenty-four isolates werearsenic-sensitive and cadmium-resistant (25.3%), 18 wereresistant to arsenic and sensitive to cadmium (18.9%), andseven were resistant to both heavy metals (7.4%).

PCR grouping results

Three PCR groups were identified in the 95 L. monocytogenesisolates recovered from sporadic human listeriosis cases inPortugal. Most isolates were of PCR group IVb (includingserotypes 4b or 4d or 4e) 71.6% (n=68): 17.9% (n=17) and10.5% (n=10) of the isolates showed PCR group IIb(including serotypes 1/2b or 3b) and PCR group IIa(including serotypes 1/2a or 3a), respectively. The D-valueobtained from these PCR grouping results was 0.449.

Combining the results from PCR grouping and sensitivityto heavy metals, it was possible to segregate the 95 isolatesinto nine groups (D=0.831). Two groups predominate: PCRgroup IVb, As- and Cd-sensitive (30.5%); PCR group IIb,As-resistant and Cd-sensitive (17.9%).

PFGE results

PFGE typing revealed a total of 39 AscI and 50 ApaImacrorestriction types among clinical isolates, distin-guished by one or more band differences ranging inmolecular size from 50 to 500 kb (Fig. 1). Combinedanalyses of AscI and ApaI PFGE data yielded a total of58 PFGE pulsotypes, with D=0.966 for AscI, D=0.979 forApaI, and D=0.986 for combined AscI and ApaI.

Comparisons based on such combined patterns arereported in Table 1 and the corresponding dendrogram inFig. 1.

Pulsotypes DD, EE, FF, G, HHb, Jb, KKa, La, LLa,LLb, Me, MM, Pb, S, T, U, V, and Ya contained two ormore strains which remained indistinguishable from eachother. Only the pulsotypes FF, LLb, MM, Pb, T, U, V, andYa contained indistinguishable strains by Cd and Assensitivity.

Pulsotypes DD, EE, FF, HHb, Jb, KKa, La, LLa, LLb,Me, MM, Pb, S, and T were constituted of L. monocyto-genes isolates from geno-serotype IVb, pulsotypes U, V, andYa of geno-serotype IIb, and pulsotype G of geno-serotypeIIa.

While strains of pulsotypes DD, FF, HHb, La, LLa, LLb,MM, Pb, S, T, V, and Ya were recovered from differentyears and geographical distribution, pulsotypes EE, G, Jb,KKa, Me, and U were related in time or geographicaldistribution. Two pulsotypes (U and KKa) have sixindistinguishable strains. Three pulsotype U strains wereisolated in the Lisbon region, respectively in February2004, August 2004, and October 2004, with the same Cdand As sensitivity. Two pulsotype KKa strains having thesame Cd and As sensitivity were isolated in March and inApril 2004.

Pulsotype G contained two indistinguishable strains butwith distinct Cd and As sensitivity isolated in February andMay 2006 in different towns.

Concerning pulsotype EE, two indistinguishable strainsbut with distinct Cd and As sensitivity were isolated indifferent hospitals in Porto in 2007 six months apart, from awoman and a newborn child.

The pulsotype Jb contained three indistinguishablestrains isolated in Lisbon in March 2006, September2006, and June 2007. Pulsotype Me is constituted by fourindistinguishable strains, three of which were isolated in thePorto region over a period of one year.

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Table 1 Listeria monocytogenes strains used in this study and collected epidemiological data concerning the cases with which they wereassociated

Isolate Hospital localization Year of isolation Clinical forma Patientgender/ageb

PCR group(geno-serotype)

Susceptibilityto As/Cdc

PFGE combined type

U I

771 Porto 2003 M Un / < 1 month IVb S/S S779 Porto 2000 NM F / 25 IVb S/R HHa780 Porto 1996 NM M / Un IVb S/S HHb781 Porto 2000 M Un / < 1 month IVb S/R HHb783 Porto 1994 M Un / < 1 month IVb R/S S784 Porto 1999 M Un / < 1 month IVb R/R Ma856 Chaves 2003 NM M / 50 IVb R/S Ka866 Matosinhos 2003 NM M / 67 IVb S/S R908 Chaves 2003 NM M / 48 IVb R/R HHb999 Coimbra 2000 NM M / 48 IIb S/S Z1001 Porto 2004 NM M / 65 IVb R/R KKa1002 Almada 1998 NM M / 54 IIb S/S NN1003 Almada 2003 NM F / 85 IVb S/S LLa1037 Chaves 2004 NM F / 74 IVb S/S Pd1059 Matosinhos 2004 NM F / 73 IVb S/R HHb1062 Lisbon 2004 NM M / 54 IIb S/S W1063 Lisbon 2002 NM M / Un IVb S/S FF1065 Lisbon 2004 NM M / Un IIb S/S U1198 Matosinhos 2004 NM M / 74 IIa S/S H1239 Matosinhos 2004 M Un / < 1 month IVb R/S BB1240 Almada 2004 M F / < 1 month IIb S/S U1241 Coimbra 2004 NM M / 44 IVb R/R KKa1242 Lisbon 2004 NM M / 57 IIb S/S U1244 Lisbon 2004 NM M / 46 IVb R/S N1348 Almada 2005 NM F / 77 IIb S/S U1383A Lisbon 2005 NM M / 40 IVb R/R JJ1541 Coimbra 2000 NM Un / Un IVb S/S LLb1542 Coimbra 2000 NM Un / Un IVb S/R HHc1543 Coimbra 2000 NM Un / Un IVb S/S Ja1544 Coimbra 2000 NM Un / Un IVb S/S DD1545 Coimbra 2003 M F / 31 IIb S/S Ya1546 Coimbra 2001 NM M / 41 IVb S/S Pc1547 Coimbra 2001 NM M / 85 IIb S/S U1548 Coimbra 1999 NM Un / Un IVb S/S KKa1549 Coimbra 1999 NM Un / Un IVb S/R MM1550 Coimbra 1997 NM Un / Un IVb S/S LLc1551 Coimbra 1997 NM Un / Un IVb S/S T1552 Coimbra 1997 NM Un / Un IVb S/S Md1553 Coimbra 1998 NM Un / Un IVb R/S La1554 Coimbra 1999 NM Un / Un IVb S/R KKa1555 Coimbra 2003 NM F / 74 IVb S/S T1556 Coimbra 2005 NM F / 56 IVb S/S Pb1562/1 Lisbon 2005 NM M / 81 IIb S/R Yb1621/1 Lisbon 2005 NM F / 72 IVb S/S LLb1761 Coimbra 2005 NM M / 57 IIb S/R A1762 Coimbra 2005 M F / 33 IIb S/R V1763 Coimbra 2005 NM M / 51 IVb R/S Mf1764 Coimbra 2005 M Un / < 1 month IVb S/R KKc1765 Coimbra 2005 M F / 31 IVb S/S Kb1766 Braga 2005 NM F / 4 months IVb S/R Me1767 Braga 2005 NM F / 46 IVb S/R KKa1768 Matosinhos 2006 M F / 31 IVb R/S Me1792 Lisbon 2006 NM M / 34 IVb R/S Jb1796 Lisbon 2006 NM F / 80 IIb S/R V

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Table 1 (continued)

Isolate Hospital localization Year of isolation Clinical forma Patientgender/ageb

PCR group(geno-serotype)

Susceptibilityto As/Cdc

PFGE combined type

U I

1803 Matosinhos 2006 NM M / 77 IVb R/S Lb1807/1 Lisboan 2006 NM F / 68 IVb S/R KKa1878 Lisbon 2006 NM M / 70 IVb S/S LLa1879 Lisbon 2006 NM M / 58 IVb R/R Jb1880 Porto 2006 NM M / 78 IIa S/S C1891 Braga 2006 NM M / 60 IIa S/S F1999 Lisbon 2006 NM M / 70 IVb S/S KKb2029 Lisbon 2007 NM F / 81 IIa S/R D2064 Matosinhos 2006 NM M / 63 IVb R/S La2065 Matosinhos 2007 NM F / 79 IIb S/R X2074 Porto 2007 M Un / < 1 month IVb S/S EE2076 Lisbon 2007 NM F / 50 IIb S/S Ya2085 Lisbon 2007 NM F / 73 IVb S/S Pa2086 Matosinhos 2007 NM M / 84 IIa S/S B2087 Lisbon 2007 NM M / 50 IVb S/R Mc2088 Porto 2006 NM M / 77 IVb S/S FF2090 Porto 2006 NM M / 54 IVb S/R Me2091 Porto 2006 NM M / 61 IIa R/S G2092 Braga 2007 NM M / 78 IVb R/S Mb2093 Braga 2007 NM M / 77 IVb R/R GG2094 Funchal 2007 NM F / 53 IVb S/S FF2095 Almada 2007 NM F / 88 IIb S/S Q2096 Porto 2005 NM M / 40 IVb S/S La2097 Coimbra 2006 NM F / 81 IVb R/S DD2098 Coimbra 2006 NM M / 83 IIb S/R AA2099 Coimbra 2006 NM M / 53 IIa S/S G2100 Coimbra 2006 NM F / 70 IVb S/S FF2101 Coimbra 2007 NM M / 66 IVb S/S II2102 Lisbon 2007 NM M / 74 IVb R/S Jb2103 Lisbon 2007 NM M / 19 IIa S/S E2104 Coimbra 2006 NM F / 68 IVb S/R MM2105 Coimbra 2007 NM M / 43 IVb S/R MM2117 Braga 2007 NM M / 76 IVa S/S CC2121 Braga 2007 NM M / 34 IIb S/S U2122 Lisbon 2007 NM M / 88 IVb S/S Pb2125 Funchal 2007 NM M / 58 IVb R/S EE2135 Lisbon 2007 NM M / 52 IIa S/R I2252 Porto 2007 NM M / 50 IVb R/S Me2253 Porto 2007 NM M / 60 IVb R/S DD2254 Porto 2007 NM M / 69 IVb S/S O2255 Porto 2007 NM F / 26 IVa R/S EE

Strains not similar at more than 98% by PFGE typing with AscI were addressed as unique, ascribed a capital letter, and in the column U (unique).Strains similar at equal to or more than 98% from at least one other isolate were listed in column I (indistinguishable) and were further typed withApaI. Strains similar at more than 98% by AscI but not similar at more than 98% by ApaI were denoted, in addition to the capital letters, bylowercase letters. So, a combined type from PCR grouping as well as from PFGE typing was determined. Strains distinguishable by their PCRgrouping or strains not similar at more than 98% by PFGE typing with AscI and ApaI were again listed as unique (U). Strains not distinguishableby their PCR grouping or strains similar at equal to or more than 98% by PFGE typing with AscI and/or ApaI were again listed asindistinguishable (I)aM: maternal/neonatal; NM, non-maternal/neonatalb Un: unknownc R: resistant; S: sensitive

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PFGE-ApaI+PFGE-AscI

apa_asc PFGE-AscI PFGE-ApaI

100

90807060 Isolate code Local

2029 Lisboa

Date

01/20072029

2103

Lisboa

Lisboa

01/2007

06/2007

1891

2091

Braga 12/2006

05/20062091

2099

05/2006

02/20062099

1198

02/2006

07/2004IIa

2135

1880

Lisboa 11/2007

09/20061880

2086

Porto

Porto

Porto

09/2006

05/2007

1761 01/2005

1348

2121

Almada

Braga

02/2005

07/20072121

1065 Lisboa

07/2007

02/2004

1547 x2/2001

1240

1242

Almada

Lisboa

10/2004

08/20041242

1002

Lisboa

Almada

08/2004

x1/1998

1762 01/2005

/IIb 1796

1062

Lisboa

Lisboa

05/2006

05/20041062

866

Lisboa 05/2004

11/2003

2098

1562 Li b

02/2006

03/20051562

999

Lisboa 03/2005

x1/2000999

2076 Lisboa

x1/2000

03/2007

1545

2065

Coimbra

Coimbra

Coimbra

Coimbra

Coimbra

Coimbra

Coimbra

03/2003

03/20072065

2102Matosinhos

Coimbra

Coimbra

Coimbra

Coimbra

Matosinhos

Matosinhos

Matosinhos

Matosinhos

Matosinhos

Matosinhos

Lisboa

03/2007

06/20072102

1792

Lisboa

Lisboa

06/2007

03/2006

1879

1543

Lisboa

Coimbra

09/2006

x5/20001543

856

Coimbra

Chaves

x5/2000

10/2003

1765 01/2005

1552

2087 Lisboa

x3/1997

04/20072087

784

Lisboa 04/2007

4/1999

2092 Braga 10/2007

1766

1768

Braga 01/2005

01/20061768

2090 Porto

01/2006

04/2006

2252

1763

Porto 05/2007

01/20051763

1553

01/2005

x2/19981553

2064

x2/1998

12/2006

2096

1803

Porto 05/2005

07/20061803

771 Porto

07/2006

04/2003771

783

Porto

Porto

04/2003

xx/1994

1551

1555

Coimbra

Coimbra

x2/1997

02/2003

Fig. 1 Dendrogram for Listeria monocytogenes pulsotypes of all 95 isolates obtained from clinical cases, Portugal, 1994–2007

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1555 Coimbra 02/2003

2095 Almada

LisboaCoimbra

Coimbra

Coimbra

Coimbra

Coimbra

CoimbraCoimbra

Coimbra

Coimbra

Funchal

Funchal

05/2007

1244

1544

05/2004

x7/20001544

2097

x7/2000

10/2006

2253

2125

Porto 12/2007

10/20072125

2074 Porto

10/2007

04/20072074

2255

Porto

Porto

04/2007

10/2007

2117

1239

Braga

Matosinhos

09/2007

09/20041239

1037

Matosinhos

Chaves

09/2004

04/2004

2254 Porto 11/2007

1556

2122 Lisboa

01/2005

08/20072122

2085

Lisboa

Lisboa

08/2007

05/2007

1546

780

Coimbra

P t

x1/2001

1/1996780

781

Porto

Porto

x1/1996

x3/2000781

908

Porto

Chaves

x3/2000

11/2003

1059

1542

Matosinhos 04/2004

x6/20001542

2088 Porto

x6/2000

05/2006IVb

1063 Lisboa x1/2002

2094

2100

04/2007

02/20062100

779 Porto

02/2006

x2/2000

2093

2101

Braga 06/2007

01/20072101

1241

01/2007

04/20041241

1548

04/2004

x1/1999

1554

1767 Braga

x2/1999

01/20051767

1001

Braga

Porto

01/2005

03/2004

1807 Lisboa 07/2006

1764

1878

Coimbra

Lisboa

01/2005

09/20061878

1003

Lisboa

Almada

09/2006

07/2003

1621

1541

Lisboa

C i b

07/2005

4/20001541

2104

Coimbra

Coimbra

x4/2000

09/20062104

2105

Coimbra

Coimbra

09/2006

03/2007

1549

1550

Coimbra

Coimbra

3/1999

09/19971550

1999

Coimbra

Lisboa

09/1997

08/2006

1383A Lisboa 02/2005

Fig. 1 (continued)

Eur J Clin Microbiol Infect Dis

Comparison with the PFGE pulsotypes database of Frenchhuman strains underlined that pulsotypes Me, Jb, Pb, HHb,FF, MM, and LLb have been previously described in humanclusters in France and are part of the main pulsotypes fromhuman strains observed in France. Pulsotypes MM and LLbare indistinguishable from pulsotypes associated with themain outbreak of listeriosis in France in 1992. The mainpulsotypes observed in Portugal, U and KKa, have not beenrecorded for French human cases.

Combination of biotyping, PCR grouping, and PFGEresults

The highest D-value, 0.994, and, thus, the greatest numberof distinguishable types (72) was obtained when theresults of multiplex PCR, sensitivity to heavy metals,and analysis of AscI and ApaI were combined.

Discussion

The 2007 Annual Community Summary Report by theEuropean Food Safety Authority (EFSA) and the EuropeanCentre for Disease Prevention andControl (ECDC) establishedan overall incidence rate of listeriosis in Europe of 3 cases permillion inhabitants, with a range of 0–11 cases per millioninhabitants. The results presented here estimated an incidencerate of listeriosis in Portugal, based on information fromvoluntary reporting, of 2.3 cases per million inhabitants for theyear 2007, which is comparable to the reported incidence inAustria, Estonia, Latvia, Slovakia, and Slovenia [17]. InPortugal’s only neighboring country, Spain, the incidencefor 2007 was similar to that described in this study and wasalso based on the voluntary reporting of cases to the NationalReference Laboratory. However, it has been demonstratedthat the implementation of an active surveillance system forlisteriosis in Navarra, a region of northern Spain, led to anincidence for this region similar to that described in countrieswith mandatory notification of listeriosis, such as France,Denmark, and the Netherlands [18]. An apparent increase inthe numbers of cases of listeriosis in recent years, observed insome other countries, e.g., Denmark, Finland, France,Germany, the Netherlands, Switzerland, and United Kingdom[3, 4], was also observed in Portugal. These results indicatethat voluntary reporting underestimates the occurrence oflisteriosis in a country, and that the EC requirement for thestatutory reporting of listeriosis will probably lead to manymore cases being uncovered. A statutory reporting andinvestigation system in Portugal will inevitably take sometime to establish, and it is recommended that, in parallel, asurveillance system for the pathogen in typical foods shouldbe established, with the capability for in-depth characteriza-tion of isolates for comparisons of clinical and food strains.

The majority of patients with listeriosis in Portugalwere people older than 60 years with bacteremia, asrecorded in some other countries, including France andUnited Kingdom [19, 20]. Most of these cases werecaused by PCR group IVb strains, which are generallyassociated with epidemic clones [21]. As in othercountries [18, 20], serogroup 1/2a became more commonthan serogroup 1/2b in 2006 and 2007.

The results of our study confirm that PCR groupingalone has the lowest discriminatory power but, whencombined with PFGE AscI and ApaI, it becomes the mostpowerful of all [22]. Nevertheless, during outbreaks, thescreening method of strains based on PCR grouping ratherthan classical serotyping is useful. Biotyping by As and Cdsensitivity gave additional information data but not enoughto consider the use of this method in a routine system ofmicrobiological surveillance. As observed in Belgium [23],an increase in strains resistant to Cd but sensitive to As wasverified.

In our study, it was possible to link sporadic cases on sixoccasions related in time and geographical distributions; thus,a common source of contamination could be suspected. Thissuspicion underlined the need to characterize food andenvironmental strains at the same time as human strains at anational level in order to trace sources of contamination and todetect and confirm related cases.

The finding that combined pulsotypes of Portugueseclinical isolates have already been described in humanclusters, and even outbreaks, in France can be explained byboth human travel and food commerce. It can be argued thatthere is, at least, a European distribution of clinical strains thatnecessitates European tracing, such as a PulseNet initiative forthe continent. Nevertheless, combined pulsotypes U and KKahave been detected in Portugal but not in France, whichsuggests that indigenous clones of L. monocytogenes mayexist in the country which might, at some time, becomeepidemic clones.

The possible epidemiological links with human strainsisolated in other countries underlines the need for an activesurveillance system of listeriosis in Portugal harmonizedwith other national systems. As the French NationalSurveillance System [20, 24], this should be based on theobligatory notification of human cases of listeriosis andepidemiological data collection, on the basis of Europeanregulation EC 178/2002 (obligatory notification of unsatis-factory food according to microbiological criteria definedby European regulation), and also on the dispatch ofisolates to a national reference laboratory for typing.

Acknowledgments This work was supported by FCT projectPTDC/AGR-ALI/64662/2006, “Listeria monocytogenes in foods:contributing data for risk assessment”.

Gonçalo Almeida was the recipient of a Fundação CalousteGulbenkian short visit grant 96780.

Eur J Clin Microbiol Infect Dis

Grateful acknowledgment goes to Laboratoire des Listeria, CentreNational de Référence des Listeria, WHO Collaborating Center forFoodborne Listeriosis, Institut Pasteur for accepting Gonçalo Almeidaand PFGE typing the strains of this work.

Members of the Research Team Ana Florinda (Centro Hospitalarde Coimbra), Graça Ribeiro and Luisa Boaventura (Hospitais daUniversidade de Coimbra), Teresa Afonso (Hospital Central doFunchal do Serviço de Saúde da Região Autónoma da Madeira),Helena Peres, Teresa Pina, and Maria José Silvestre (Hospital CurryCabral, Lisboa), Maria Dolores Pinheiro (Hospital de São João,Porto), Maria Alberta Faustino and Maria Carmen Iglesias (Hospitalde São Marcos, Braga), José Diogo, Ana Rodrigues, and IsabelNascimento (Hospital Garcia da Horta, Almada), Fernanda Bessa andElmano Ramalheira (Hospital Infante D. Pedro-Aveiro, E.P.E.), JoãoLago (Hospital Militar de Belém, Lisboa), Maria Antónia Read(Hospital Pedro Hispano, Matosinhos), Lurdes Monteiro, LuísMarques Lito, and J. Melo Cristino (Hospital Santa Maria, Lisboa),Maria Helena Ramos (Hospital Santo António, Porto), Maria AugustaGuimarães (Instituto Português de Oncologia, Porto).

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