Research in Microbiology 158 (2007) 10e17www.elsevier.com/locate/resmic

Evaluation of a modified single-enzyme amplified fragment lengthpolymorphism (SE-AFLP) technique for subtyping Salmonella enterica

serotype Enteritidis

Giovanni M. Giammanco a,*, Caterina Mammina a, Cristina Romani b, Ida Luzzi c,Anna Maria Dionisi c, Antonino Nastasi b

a Dipartimento di Igiene e Microbiologia ‘‘G. D’Alessandro’’, Universita degli Studi di Palermo, via del Vespro 133, 90127 Palermo, Italyb Dipartimento di Sanita Pubblica ‘‘G.B. Morgagni’’, Universita degli Studi di Firenze, Firenze, Italy

c Dipartimento di Malattie Infettive, Parassitarie e Immunomediate, Istituto Superiore di Sanita, Rome, Italy

Received 10 May 2006; accepted 9 October 2006

Available online 19 December 2006

Abstract

Salmonella enterica subsp. enterica serotype Enteritidis is not readily subtyped beyond the level of phage type (PT). Pulsed field gel elec-trophoresis (PFGE) is generally acknowledged to be the most discriminating typing method for Salmonella, but only a restricted variety of PFGEtypes has been described for S. enterica serotype Enteritidis. In the present study, a modification of the SE-AFLP typing method was used toinvestigate both outbreak and apparently sporadic isolates of S. enterica serotype Enteritidis belonging to different PTs and/or PFGE types. Themethod proved to be as discriminatory as PFGE when combined with phage typing, and provided subtyping data consistent with epidemiologicalinformation. Although the modified SE-AFLP typing method did not prove to achieve a superior discriminatory ability in resolving clusters, ithas a high enough throughput for use in outbreak investigations. This method can be used in combination with other typing methods to obtainepidemiologically relevant subtyping data on S. enterica serotype Enteritidis.� 2006 Elsevier Masson SAS. All rights reserved.

Keywords: Salmonella enterica serotype Enteritidis; Typing; Amplified fragment length polymorphism; Pulsed field gel electrophoresis

1. Introduction

Salmonella enterica serotype Enteritidis remains the mostprevalent serotype infecting humans worldwide [5,8]. TheWorld Health Organization Global Salm-Surv network re-ported that in the period 2000e2002, S. enterica serotype En-teritidis was by far the most common serotype reported fromhuman sources worldwide, accounting for 65% of isolates[5,8]. In Europe, during those same years S. enterica serotypeEnteritidis accounted for 79e84% of human isolates [10]. En-ter-Net data confirmed the ranking, but at a lower proportion:

* Corresponding author. Tel.: þ39 091 655 3663; fax: þ39 091 616 0711.

E-mail address: g.m.giammanco@unipa.it (G.M. Giammanco).

0923-2508/$ - see front matter � 2006 Elsevier Masson SAS. All rights reserv

doi:10.1016/j.resmic.2006.10.004

68e71% [3,10]. Indeed, in many European countries includ-ing Italy, after a phase of decline between the late 1990s and2002 due to ongoing implementation of more stringent Euro-pean food safety legislation in breeding flocks and improvedsanitation and farming practices for egg-layer and broilerflocks, the incidence of human infection has once again in-creased starting during the third quarter of 2002 [5,10].

Phage typing has traditionally been the first-line method forcharacterizing serotype Enteritidis isolates, but the predomi-nance of a few phage types (PTs) and sporadic occurrenceof inconsistent findings due to phage conversion have madeit necessary to adopt molecular typing approaches[2,6,11,12,14,15,17]. Pulsed field gel electrophoresis (PFGE)is currently the gold standard method for epidemiological sub-typing of Salmonella strains, and laboratory networks like

ed.

11G.M. Giammanco et al. / Research in Microbiology 158 (2007) 10e17

PulseNet in the USA and Salm-gene in Europe provide stan-dard operating procedures [19]. Unfortunately, although a largevariety of pulsed-field types could be obtained for S. entericaserotype Enteritidis, most often a very limited number of com-mon types accounts for the large majority of total isolates [6].Therefore, the use of other molecular subtyping methodsshould be considered for S. enterica serotype Enteritidis.The range of DNA fingerprinting techniques has been progres-sively expanding in recent years to include several polymerasechain reaction (PCR)-based approaches, such as amplifiedfragment length polymorphism analysis (AFLP). The lattertechnique, originally developed as a universal DNA typingmethod for crop plants, has been widely used in the last tenyears for characterization of plant, animal and prokaryoticDNAs [1].

AFLP is a PCR-based DNA fingerprinting methodologywhich includes two steps: endonuclease restriction of theDNA of the strain under examination, and selective amplifica-tion of a subset of DNA fragments achieved by the ligation ofoligonucleotide adapters and subsequent annealing of primerscontaining adapter-targeted sequences plus one arbitrarynucleotide as the final 30 base. The classical single enzymeapproach (SE-AFLP), previously employed in epidemiologicalanalysis of Legionella and Helicobacter species [4,7], requiresseparate assessment in order to be suitable for discriminationof the four selective primers (ending with A, C, G or T).When SE-AFLP was applied to S. typhimurium strains, onlyprimer -C was able to produce reasonably well defined anddiscriminative banding patterns, while the other primers pro-duced less distinct patterns, with either too many or too fewbands, making comparative analysis difficult [18]. SE-AFLPhas already been applied to sporadic and epidemic S. entericaserotype Enteritidis isolates with promising results [2,15], butthe need for a hierarchical assessment of the usefulness ofeach primer makes this classical SE-AFLP approach cumber-some. A modified SE-AFLP protocol using a mix of the fourprimers differing by the final 30 base in the same PCR ampli-fication tube has been developed in our laboratory [12]. Whenthis modified 4-primer SE-AFLP protocol was used for sub-typing a small set of representative S. enterica serotype Enter-itidis strains, it enabled more complex and discriminativepatterns of bands to be obtained compared to the classical sin-gle-primer PCR reactions. Primers -A, -C and -T showed poordiscriminatory power when used alone, producing one or twobands each, while primer -G was unable to produce amplifica-tion products from any of the S. enterica serotype Enteritidisisolates except for one. When the same set of strains wassubmitted to PCR by using a mix of the four primers, morecomplex electrophoretic patterns, including five to sevenbands, were obtained [12].

In this study, this modified 4-primer SE-AFLP protocol hasbeen applied to a large number of apparently sporadic serotypeEnteritidis isolates and to a small group of epidemic clustersidentified during a 13-month period in 2003e2004 at the Cen-ter for Enteric Pathogens of the Italian National Institute ofHealth (ISS). The results of this modified SE-AFLP typingwere compared to phage typing, resisto-typing and PFGE

typing and analyzed on the basis of the epidemiological infor-mation available for the isolates, in order to evaluate the use-fulness of this new method for epidemiological typing andoutbreak investigation.

2. Materials and methods

2.1. Bacterial strains

A total of 185 strains of serotype Enteritidis isolated inItaly during 2003e2004 were used in this study. One-hundredand sixty-six isolates were from apparently sporadic humancases of salmonellosis, and 19 were from five different out-breaks and included both human and food isolates. All isolateswere serotyped using the KauffmanneWhite scheme [16].

2.2. Phage typing

Phage typing was performed by using standard methodsand phages provided by the International Phage typing Ref-erence Laboratory (Health Protection Agency, Colindale,London, UK) [20].

2.3. Antimicrobial susceptibility testing

Susceptibility patterns were determined by the disk diffu-sion method following the National Committee for ClinicalLaboratory Standards, NCCLS (now Clinical and LaboratoryStandards Institute, CLSI) guidelines [13]. The followingantimicrobial disks (Oxoid, Basingstoke, UK) were used:ampicillin (10 mg), cefotaxime (30 mg), ciprofloxacin (5 mg),chloramphenicol (30 mg), gentamicin (10 mg), kanamicin(30 mg), nalidixic acid (30 mg), streptomycin (10 mg), sulfon-amides (300 mg), tetracycline (30 mg), and trimethoprim(5 mg). Escherichia coli ATCC 25922 was used as the suscep-tible control strain.

2.4. Molecular typing

2.4.1. Pulsed field gel electrophoresisThe preparation of genomic DNA of serotype Enteritidis

isolates was performed according to Salm-gene protocol [14].Briefly, this involved proteinase K lysis of cells and a seriesof washes at 50 �C followed by digestion with 50 U XbaI(New England Biolabs, Beverly, MA) for 18 h at 37 �C.DNA macrorestriction fragments were resolved on 1% pulsedfield certified� agarose (Bio-Rad Laboratories, Hercules, CA)gels on a CHEF-DRII system (Bio-Rad Laboratories), with theS. enterica serotype Braenderup reference strain H9812(kindly supplied by PulseNet US, CDC) as the molecular ref-erence marker [19]. The XbaI fragments were separated byelectrophoresis in 0.5� TBE for 22 h at 4 �C with 2 to 64 sof linear ramping at 6 V/cm. The gels were stained for10 min in ethidium bromide solution (0.4 mg/ml), destainedin distilled water for 20 min and photographed under UV light.The images were stored as a tag image file format (TIFF) file.

12 G.M. Giammanco et al. / Research in Microbiology 158 (2007) 10e17

TIFF files were analyzed with BioNumerics softwareversion 3.5 (Applied Maths, Kortrijik, Belgium). The softwareallowed us the assignation of a PFGE profile to each S. entericaserotype Enteritidis pattern, comparing it with the profiles li-brary created by the Salm-gene project. Comparison betweenprofiles was made using algorithms available within the pro-gram and a dendrogram was produced using the Dice coeffi-cient and the unweighted pair-group method (UPGMA) witha 1.5% tolerance limit and 1.5% optimization [6].

2.4.2. SE-AFLPAfter extraction, DNA was submitted to a simultaneous re-

action of digestion with the restriction endonuclease HindIIIand ligation at 37 �C for 3e4 h. Adapters H1, 50-ACGG-TATGCCACAG-30 and H2, 50- AGCTCTGTCGCATACCGT-GAG-30 were used. Digested-ligated DNA was heated to80 �C for 10 min to inactivate the ligase, and subsequentPCR was carried out in 50 ml amplification mixture containing200 mM each deoxynucleoside triphosphate (dNTP), 2.5 mMMgCl2, 150 ng of each primer and 1 U of Taq polymerase.A mix of four primers was used for PCR reactions. Theprimers had the sequence 50-GGTATGCGACAGAGCTTX-30, where X was either A, T, G or C, respectively. PCR ampli-fication consisted of 1 cycle at 94 �C for 4 min, followed by 33cycles of 94 �C for 1 min, 60 �C for 1 min, and 72 �C for2.5 min. PCR products were detected on a 1.5% agarose gelrun alongside a 100 bp ladder at 100 V for 3 h, followed bystaining in ethidium bromide. Photos of the gels were takenunder UV light and the gel images were captured in imagefiles (TIFF). SE-AFLP profiles were detected by RestrictoScansoftware (Taxotron package, Taxolab, Institut Pasteur, Paris,France) and the molecular weight of the bands was calculatedby comparing migration values to the average of the two near-est molecular weight standard lanes. SE-AFLP banding pat-terns were analyzed by the Taxotron software (Taxolab). Adistance coefficient was calculated using the complement ofthe Dice index with an allowed error limit of 5% and dendro-grams were generated by the UPGMA algorithm.

2.5. Evaluation of discriminatory power

In order to assess the discriminatory power of the differenttyping methods for characterizing S. enterica serotype Enteri-tidis isolates, the numerical Index of Discrimination proposedby Hunter and Gaston was calculated [9].

3. Results

The results obtained by the four typing methods used onsporadic and epidemic isolates are summarized in Tables 1and 2, respectively.

Phage typing differentiated 153 sporadic isolates into 20types: the most common were phage type (PT) 4, PT21, PT1and PT14b, which were found in 43 (28.1%), 28 (18.3%),22 (14.4%) and 13 (8.5%) isolates, respectively. PT1c andPT2 were found in eight (5.2%) isolates each and PT8 in 7(4.6%), while the remaining types appeared only in one, two

or three strains. Among the 15 RDNC strains, nine could betyped according to their phage susceptibility pattern into threedifferent groups including two, three and four isolates, respec-tively. Six strains were untypable (Table 1).

When resistance patterns were analyzed, 11 R-types couldbe described among 165 sporadic isolates, but 118 (71.6%) ofthem were susceptible to all the antibiotics tested. The mostfrequent resistance pattern was represented by the single resis-tance to sulfonamides (10.3%), followed by the single resis-tance to nalidixic acid (5.5%) and ampicillin (4.2%), whilemore complex patterns including two or more (up to four)antibiotics were observed in no more than four isolates perpattern (Table 1).

Molecular characterization of 166 isolates by XbaI PFGEanalysis identified within the Salm-gene profiles library 11pulsotypes among 155 sporadic isolates, each displaying 11to 16 bands restriction patterns and differing one to ten bandsfrom each other (Fig. 1). Type SENTXB.0001 was found in 73(47.1%) isolates, while type SENTXB.0005 was foundin 41 (26.5%), type SENTXB.0002 in 18 (11.6%), typesSENTXB.0046 in seven (4.5%), type SENTXB.0014 in six(3.9%) and the remaining six types only in one or three iso-lates. In eleven isolates unnamed types were found that werenot included in the Salm-gene library and were labeled bya progressive number (Table 1).

The SE-AFLP protocol using a mix of the four primers (4-primer SE-AFLP) produced electrophoretic patterns includingseven to ten bands (Fig. 2). These patterns were reproduciblein individual experiments. By this modified SE-AFLP method,12 typesdA to Ndwere obtained: 105 (68.2%) of the 154sporadic isolates typed by this method were included in themost frequent type A, whereas 20 (13%) were attributed totype C, eight (5.2%) to type G, six (3.9%) to type B andone to four to the remaining eight types (Table 1).

A single 4-primer SE-AFLP pattern (type C) was consis-tently associated with strains belonging to PTs 2 and 8 andpulsotype SENTXB.0002. In contrast, many PTs wererepresented by SE-AFLP in the major cluster A, mostly corre-sponding to three different pulsotypes: SENTXB.0001,SENTXB.0005 and SENTXB.0014. Two SE-AFLP groups,B and D, included the majority of the RDNC strains, butRDNC patterns including phages 2 and 6 were associatedwith pulsotype SENTXB.0046 and, except for one isolate,SE-AFLP type B. SE-AFLP group G was mostly associatedwith antibiotic resistant strains. No other peculiar associationbetween 4-primer SE-AFLP types, pulsotypes, R-types andPTs could be described. Most interestingly, at least two 4-primer SE-AFLP types could be associated with each of theseven PFGE types including more than one isolate. In partic-ular, 66 isolates showing the most frequent SENTXB.0001PFGE pattern belonged to seven different SE-AFLP types,with type A being the most frequently represented (52 iso-lates). The SENTXB.0002 profile could be subdivided intothree different SE-AFLP types (A, C and D), with type C ac-counting for 14 out of 17 of them, while SENTXB.0005 wasmore homogeneous, since only one out of 40 isolates belongedto an SE-AFLP type other than A. The less frequent SENTXB

13G.M. Giammanco et al. / Research in Microbiology 158 (2007) 10e17

Table 1

Typing of 166 Salmonella serotype Enteritidis strains from sporadic cases of

salmonellosis

Phage

type

XbaI PFGE

type

SE-AFLP

type

R-type No. of

isolates

1 SENTXB.0001 A Susceptible 1

1 SENTXB.0001 A Na 8

1 SENTXB.0001 A Su, Na 1

1 SENTXB.0001 G Su, Na 1

1 SENTXB.0001 NA Susceptible 1

1 SENTXB.0002 C Susceptible 1

1 SENTXB.0005 A Susceptible 7

1 SENTXB.0014 A Su, Na 1

1 Unnamed-1 G T 1

1C SENTXB.0001 A Susceptible 1

1C SENTXB.0005 A Susceptible 7

2 SENTXB.0001 A Susceptible 1

2 SENTXB.0002 C Susceptible 6

2 SENTXB.0002 NA Su 1

4 SENTXB.0001 A Susceptible 24

4 SENTXB.0001 A Su 2

4 SENTXB.0001 C Susceptible 1

4 SENTXB.0001 F Susceptible 1

4 SENTXB.0001 G S, Su 2

4 SENTXB.0001 G NA 1

4 SENTXB.0001 I Susceptible 1

4 SENTXB.0001 NA Susceptible 4

4 SENTXB.0002 A Susceptible 1

4 SENTXB.0005 A Susceptible 1

4 SENTXB.0014 A Susceptible 1

4 SENTXB.0014 A Su 1

4 Unnamed-2 A Susceptible 1

4 Unnamed-3 A Susceptible 1

4 Unnamed-4 N Susceptible 1

4B SENTXB.0001 A Susceptible 1

4B SENTXB.0005 A Susceptible 1

5A SENTXB.0007 A A 1

6 SENTXB.0001 A Na 1

6A SENTXB.0001 A A 2

8 SENTXB.0002 C Susceptible 4

8 SENTXB.0006 C Susceptible 1

8 SENTXB.0009 C Susceptible 2

11 SENTXB.0017 C Susceptible 1

14B SENTXB.0001 A Susceptible 4

14B SENTXB.0001 A Su 1

14B SENTXB.0001 E Su 1

14B SENTXB.0001 N Su 1

14B SENTXB.0001 NA Susceptible 2

14B SENTXB.0014 A Susceptible 1

14B SENTXB.0014 H Susceptible 1

14B SENTXB.0046 B Su 1

14B Unnamed-5 NA Susceptible 1

21 SENTXB.0001 E Su, Na 1

21 SENTXB.0005 A Susceptible 15

21 SENTXB.0005 A A 1

21 SENTXB.0005 A Su 4

21 SENTXB.0005 L Susceptible 1

21 SENTXB.0005 NA Susceptible 1

21 SENTXB.0044 A Susceptible 1

21 SENTXB.0044 A Su 1

21 SENTXB.0044 I Susceptible 1

21 Unnamed-6 A Su, T, Tm 1

21 Unnamed-7 NA Susceptible 1

21C SENTXB.0005 A Susceptible 3

30 Unnamed-8 G A, Su 1

33 Unnamed-9 M A, S, Su, T 1

(continued)

types 0009, 0014, 0044 and 0046 showed two SE-AFLP typeseach. Fig. 3 shows the subtyping obtained by 4-primer SE-AFLP when applied to the seven major PFGE types.

Intra-laboratory reproducibility of the 4-primer SE-AFLPtechnique was assessed by repeated processing of the isolates.Duplicate amplification and migration was performed for 29isolates, while 19 were processed three times. Repetitions al-ways produced identical patterns.

Table 3 summarizes the results obtained by applying thediscrimination index of Hunter and Gaston [9] to the resultsobtained by the different typing methods on sporadic isolatesand allows for a comparison of their performances.

When five groups of epidemic isolates were analyzed, alltyping methods agreed in identifying a single type in threeof them (Table 2). On the contrary, in two epidemics, PFGEonly identified two types among the three strains included ineach group. A PFGE banding pattern not previously encoun-tered in sporadic strains, pattern SENTXB.0031, was foundin one of the epidemic groups. PFGE patterns discordantwith respect to the other typing methods differed by one(SENTXB.0001/14) or two (SENTXB.0001/31) bands.

4. Discussion

As S. enterica serotype Enteritidis is the most frequentlyidentified serotype from humans worldwide, it is essential toundertake subtyping to investigate and confirm outbreaks,

Table 1 (continued )

Phage

type

XbaI PFGE

type

SE-AFLP

type

R-type No. of

isolates

35 SENTXB.0001 A Susceptible 1

35 SENTXB.0014 A Susceptible 1

36 SENTXB.0001 G S, Su 1

RDNCa SENTXB.0046 A Su 1

RDNCa SENTXB.0046 B Susceptible 2

RDNCa SENTXB.0046 B Su 1

RDNCb SENTXB.0046 B Susceptible 1

RDNCb SENTXB.0046 B Su 1

RDNCc SENTXB.0002 D A 1

RDNCc SENTXB.0002 D A, Su 1

RDNCc SENTXB.0009 D A 1

RDNC SENTXB.0002 C Susceptible 1

RDNC SENTXB.0002 C Susceptible 1

RDNC SENTXB.0002 C Susceptible 1

RDNC SENTXB.0013 A Susceptible 1

RDNC Unnamed-10 C A 1

RDNC Unnamed-11 NA A, Su 1

NT SENTXB.0001 A Susceptible 1

NT SENTXB.0001 A Susceptible 1

NT SENTXB.0001 A Su 1

NT SENTXB.0001 E Susceptible 1

NT SENTXB.0001 E Susceptible 1

NT SENTXB.0001 G Susceptible 1

NA SENTXB.0001 A A, Su, Na 1

NA, not available; NT, not typable; RDNC, reacts but does not conform; Na,

nalidixic acid; A, ampicillin; S, streptomycin; Su, sulfonamides;

T, tetracycline; Tm, trimethoprim.a Phage susceptibility pattern: 2, 6.b Phage susceptibility pattern: 2, 3, 5, 6, 12, 13.c phage susceptibility pattern: 1, 4, 8, 9, 10.

14 G.M. Giammanco et al. / Research in Microbiology 158 (2007) 10e17

Table 2

Epidemiological data and typing results of 19 S. enterica serotype Enteritidis epidemic strains

Epidemica Isolate ISS code Origin Sampling date Phage type XbaI PFGE type SE-AFLP type R-type

a 46 58/1 Brindisi 02/05/2003 4 SENTXB.0031 A Susceptible

a 47 58/2 Brindisi 02/05/2003 4 SENTXB.0001 A Susceptible

a 48 58/6 Brindisi 05/05/2003 4 SENTXB.0001 A Susceptible

b 95 110/10 Brindisi 27/08/2003 8 SENTXB.0002 C Susceptible

b 96 110/12 Brindisi 27/08/2003 8 SENTXB.0002 C Susceptible

c 132 127/1 Turin 05/09/2003 4 SENTXB.0001 A Su

c 133 127/3 Turin 05/09/2003 4 SENTXB.0001 A Su

c 134 127/4 Turin 04/09/2003 4 SENTXB.0001 A Su

c 135 127/6 Turin 03/09/2003 4 SENTXB.0001 A Su

c 136 127/9 Turin 02/09/2003 4 SENTXB.0001 A Su

d 141 136/2 Brindisi 18/10/2003 14B SENTXB.0001 A Susceptible

d 142 136/3 Brindisi 17/10/2003 14B SENTXB.0001 A Susceptible

d 143 136/4 Brindisi 13/10/2003 14B SENTXB.0014 A Susceptible

e 169 20/1 Rome 19/02/2004 8 SENTXB.0002 C Susceptible

e 170 20/2 Rome 19/02/2004 8 SENTXB.0002 C Susceptible

e 171 20/3 Rome 19/02/2004 8 SENTXB.0002 C Susceptible

e 172 20/4 Rome 19/02/2004 8 SENTXB.0002 C Susceptible

e 173 20/5 Rome 19/02/2004 8 SENTXB.0002 C Susceptible

e 174 20/6 Rome 19/02/2004 8 SENTXB.0002 C Susceptible

Su, sulfonamides.a Epidemic ‘‘a’’ was related to eggs consumption; no data on the food involved in epidemics ‘‘b’’ to ‘‘e’’ are available.

accurately evaluate the trends in infection cases and identifyany emerging issues.

Phage typing has been traditionally applied to thecharacterization of Salmonella serotypes and has proven tobe an invaluable tool, mainly for long-term epidemiological

studies performed over wide geographic areas. However,a number of both technical and theoretical troubles hamperwide application of this subtyping method: among the morerelevant ones, the unavailability of phage suspensions exceptfor a limited number of reference laboratories, difficulties in

100

908070605040

% identity

SENTXB.0002

SENTXB.0006

SENTXB.0009

SENTXB.0046

SENTXB.0001

SENTXB.0007

SENTXB.0005

SENTXB.0013

SENTXB.0014

SENTXB.0044

SENTXB.0031

SENTXB.0017

1100

700

Kbp300

240

160

120

80 40

Fig. 1. Phylogenetic dendrogram produced using Dice coefficient and the unweighted pair-group method (UPGMA) with a 1.5% tolerance limit and 1.5%

optimization from the restriction patterns obtained by PFGE from 175 Salmonella serotype Enteritidis isolates using endonuclease XbaI.

15G.M. Giammanco et al. / Research in Microbiology 158 (2007) 10e17

standardization of performing and reading, negative effects onspecificity by phage conversion or adaptation of surface bacte-riophage receptors and consequent phage type shift [11,17].

The use of molecular typing has become increasinglyimportant for the characterization of Salmonella, but it hasencountered serious limitations when applied to serotypeEnteritidis, since only a few bacterial clones seem to be circu-lating worldwide [6]. In fact, PFGE, a well established method

80 84 88 92 96 100

B

A

C

D

E

F

G

H

I

L

M

N

% similaritybp500 400 300

Fig. 2. UPGMA phylogenetic dendrogram showing the genetic distances

obtained by the 4-primer SE-AFLP for the 12 patterns produced by the 173

Salmonella serotype Enteritidis isolates studied. A to N: amplification pattern

codes.

that has been largely applied to studies of S. enterica, recog-nizes only a few profiles among the large majority of circulat-ing serotype Enteritidis isolates, and most strains are attributedto two main lineages, the first containing PT1, PT4 and PT6and the second including PT2 and PT8. Moreover, PFGE isa labor-intensive and technically demanding method and re-quires a specific electrophoresis apparatus that is likely to berestricted to research and reference laboratories.

SE-AFLP is a PCR-based DNA fingerprinting methodologythat showed promising results when applied to the typing ofSalmonella serotypes [2,15,18], but it requires a cumbersomeseparate assessment of the usefulness of each of the four selec-tive primers in order for the suitability for discrimination to beassessed. Moreover, when this classical SE-AFLP approachwas applied to S. enterica serotype Enteritidis strains, it wasunable to produce discriminative patterns due to the very lim-ited number of bands obtained [12].

Thus, the purpose of our study was to evaluate a simplifiedSE-AFLP version, modified by the use of a mix of the four se-lective primers in the same PCR amplification (4-primer SE-AFLP) that had already proven to be promising when appliedto characterization of a small group of serotype Enteritidis iso-lates [12]. This new method was evaluated on a large numberof strains both in terms of discriminatory power and user-friendliness in comparison with two reference phenotypic typ-ing methods, phage-typing and resisto-typing, and with PFGE,the gold standard for the molecular typing of Salmonella.From our results, this 4-primer SE-AFLP protocol produceda limited number of subtypes from the serotype Enteritidis iso-lates analyzed, further confirming the genetic homogeneity ofthis serotype, but the typing was consistent with the availableepidemiological data. When compared to PFGE, the 4-primerSE-AFLP showed lower discriminative power, but the two

SENTXB.0044(3)

I

A67

33

SENTXB.0014(6)

A

H

83

17

SENTXB.0005(40)

A

L3

97

SENTXB.0002(17)

D A

C

12 6

82

SENTXB.0001(66)

A

G IF

E

C

N

2

78

6

2 8 2 2

SENTXB.0046(7)

B

A14

86

SENTXB.0009(3)

D

C67

33

Fig. 3. Percent distribution of the subtypes obtained by the 4-primer SE-AFLP method within each of the seven major PFGE types. The number of isolates analyzed

within every single PFGE type is shown in parentheses.

16 G.M. Giammanco et al. / Research in Microbiology 158 (2007) 10e17

molecular typing methods have approximately equal utility ifcombined with phage typing. In fact, both combinations areable to reach the minimal discrimination index of 0.90 identi-fied as the desirable threshold for results to be interpreted withconfidence. By combining 4-primer SE-AFLP typing withPFGE, a discriminatory power similar to that displayed byphage typing alone can be obtained. In particular, all of thePFGE types including more than one isolate could be furthersubtyped by 4-primer SE-AFLP, which was especially effec-tive in characterizing isolates within the major SENTXB.0001PFGE profile, while poorer discrimination was obtained withinthe SENTXB.0005 type (Fig. 3). A sharp increase in discrim-ination was also achieved by the combination of 4-primerSE-AFLP and R-typing, two methods that do not requirespecialized equipment, if compared to all typing methodswhen used alone, PFGE included. When outbreak isolateswere analyzed, 4-primer SE-AFLP performed even betterthan PFGE in clustering epidemic clones according to theirepidemiological source. A particular association between iso-lates belonging to the 4-primer SE-AFLP G type and antibioticresistance must be underlined.

Four-primer SE-AFLP is less labor-intensive than PFGE,does not require specialized equipment and minimizes thetime to results. The advantages in terms of turnaround,ease of use and equipment cost suggest that 4-primer SE-AFLP could be an attractive alternative to PFGE, mainlyfor epidemiological investigation of foodborne outbreaksand especially when real-time results may contribute to sup-porting timely control measures. An association with PFGEor other molecular methods could also be recommended inorder to achieve fine subtyping in highly homogeneousserotypes such as Enteritidis or when phage typing is notavailable. However, the limited discriminative power is anargument against the use of 4-primer SE-AFLP as a singlegenotypic method mainly for large-scale studies. Similarlyto results observed with all amplification-based typingmethods, the absence of correspondence in 4-primer SE-AFLP subtyping patterns obtained in different laboratoriescould be expected and interlaboratory reproducibility wouldneed to be checked before a database of reference patternscan be constructed.

Table 3

Index of discrimination of typing methods used to discriminate among

sporadic S. enterica serotype Enteritidis strains

Typing method No. of types Size of largest

cluster

(% of typed strains)

ID

Phage typing 20 28.1 0.855

XbaI PFGE typing 11 47.1 0.695

Modified SE-AFLP typing 12 68.2 0.516

R-typing 11 71.6 0.475

Phage þ PFGE typing 43 22.6 0.915

Phage þ SE-AFLP typing 39 20.9 0.911

Phage þ R-typing 34 24.3 0.907

SE-AFLP þ R-typing 27 50.3 0.725

SE-AFLP þ PFGE typing 29 33.8 0.813

ID, index of discrimination according to Hunter and Gaston [9].

Acknowledgments

We would like to thank all the participants in the Enter-netItalian Network for providing data and strains. We would alsolike to acknowledge the technical support of Ildo Benedetti,Sergio Arena and Slawomir Owczarek. This work was partlysupported by the Ricerca finalizzata 2003 grant ‘‘Studio e ap-plicazioni di metodi integrati medico/veterinari per la sorve-glianza delle zoonosi a trasmissione alimentare’’ from theItalian Ministry of Health.

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