Identification of plasmids by PCR-based replicon typing

www.elsevier.com/locate/jmicmeth

Journal of Microbiological Methods 6

Identification of plasmids by PCR-based replicon typing

Alessandra Carattolia,*, Alessia Bertinia, Laura Villaa, Vincenzo Falbob,

Katie L. Hopkinsc, E. John Threlfallc

aDepartment of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Rome, ItalybDepartment of Cell Biology and Neurosciences, Istituto Superiore di Sanita, Rome, Italy

cLaboratory of Enteric Pathogens, Health Protection Agency Centre for Infections, 61 Colindale Avenue, London NW9 5HT, United Kingdom

Received 2 February 2005; received in revised form 15 March 2005; accepted 17 March 2005

Available online 2 June 2005

Abstract

The epidemiological importance of tracing plasmids conferring drug resistance prompted us to develop a PCR method based

on replicons (inc/rep PCR) of the major plasmid incompatibility groups among Enterobacteriaceae. Eighteen pairs of primers

were designed to perform 5 multiplex- and 3 simplex-PCRs, recognizing FIA, FIB, FIC, HI1, HI2, I1-Ig, L/M, N, P, W, T, A/C,

K, B/O, X, Y, F, and FIIA. The specificity of the method was tested on a collection of 61 reference plasmids and on 20

Salmonella enterica strains of different serotypes isolated in Italy. Results indicated that the inc/rep PCR method demonstrates

high specificity and sensitivity in detecting replicons on reference plasmids and also revealed the presence of recurrent and

common plasmids in epidemiologically unrelated Salmonella isolates of different serotypes. These results suggest that the

method is potentially applicable to a large number of strains to trace the diffusion of specific multi-drug resistance plasmids in

different environments.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Plasmids; PCR; Replicon

1. Introduction

Plasmids are extra-chromosomal circular fragments

of DNA that replicate autonomously in a host cell.

They are present in nearly all bacterial species and

vary in size from a few to more than several hundred

0167-7012/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.mimet.2005.03.018

* Corresponding author. Tel.: +39 6 4990 3128; fax: +39 6 4938

7112.

E-mail address: [email protected] (A. Carattoli).

kilobase pairs (kb) (Waters, 1999). Plasmids appear to

increase bacterial genetic diversity, acquiring and los-

ing genes, and can be horizontally exchanged among

bacterial populations by conjugation or mobilization

(Francia et al., 2004). They contain genes essential for

initiation and control of replication and accessory

genes that may be useful to their bacterial host such

as antimicrobial resistance or virulence genes (Ama-

bile-Cuevas and Chicurel, 1992; Bergstrom et al.,

2000; Thomas, 1973).

3 (2005) 219–228

A. Carattoli et al. / Journal of Microbiological Methods 63 (2005) 219–228220

A formal scheme of plasmid classification is based

on incompatibility (Inc) groups (Novick, 1987). The

procedure for incompatibility grouping is based on the

introduction, by conjugation or transformation, of a

plasmid of an bunknownQ Inc group into a strain

carrying a plasmid of a known Inc group. If the

resident plasmid is eliminated in the progeny,

the incoming plasmid is assigned to its same Inc

group (Datta and Hedges, 1971). Plasmids with the

same replication control are bincompatibleQ, whereasplasmids with different replication controls are

bcompatibleQ. On this basis two plasmids belonging

to the same Inc group cannot be propagated in the

same cell line (Datta and Hughes, 1983; Couturier et

al., 1988). Inc group identification has been frequently

used to classify plasmids. The method has been an

important tool to trace the diffusion of plasmids con-

ferring antimicrobial resistance and also to follow

the evolution and spread of emerging plasmids

(Anderson et al., 1977).

In 1988 Couturier et al. developed a new method

for the identification of the major replicons of plas-

mids circulating among the Enterobacteriaceae.This

method was based on hybridization with 19 DNA

probes that recognize different basic replicons (Cou-

turier et al., 1988). The conjugation- and hybridiza-

tion-based methodologies cannot be easily applied to

a large number of strains and their application has

been limited by the laborious and time-consuming

work required. A PCR-based detection of plasmids

was previously devised on the basis of published

sequences but it was limited to the IncP, IncN, IncW

and IncQ plasmids (Gotz et al., 1996).

The necessity of tracing plasmids conferring drug

resistance prompted us to develop an inc/rep PCR-

based typing method. In this method, 18 pairs of

primers were designed to perform 5 multiplex- and

3 simplex-PCRs, recognizing the FIA, FIB, FIC, HI1,

HI2, I1-Ig, L/M, N, P, W, T, A/C, K, B/O, X, Y, F, and

FIIA replicons, representative of the major plasmid

incompatibility groups circulating among the Entero-

bacteriaceae (Couturier et al., 1988).

The specificity of the method was tested on 61

reference plasmids. Plasmids in a collection of 20

previously characterized multi-drug resistant Salmo-

nella enterica isolates of different serotypes (Pezzella

et al., 2004) have then been typed by PCR-based inc/

rep typing.

2. Materials and methods

2.1. Bacterial strains and plasmids

Sixty-one Escherichia coli and S. enterica strains

carrying plasmids belonging to 22 different incompat-

ibility (Inc) groups, were available from the collec-

tions of the Istituto Superiore di Sanita in Rome and

the UK Health Protection Agency. Thirty-nine plas-

mids analyzed in this study were reference plasmids

of known Inc groups (Anderson et al., 1977; Carattoli

et al., 2001; Couturier et al., 1988; Frydman and

Meynell, 1969; Grindley et al., 1972; Hedges and

Datta, 1971;Villa et al., 2000); 22 plasmids were

assigned to Inc groups by conjugation with reference

plasmids (Datta and Hedges, 1971), or by hybridiza-

tion with replicon probes (Couturier et al., 1988).

Twenty S. enterica among the 58 isolates previously

characterized (Pezzella et al., 2004), isolated in Italy

from animals and food of animal origin at the Istituto

Zooprofilattico Sperimentale delle Venezie and at the

Istituto Zooprofilattico Sperimentale dell’Abruzzo e

Molise in 2000–2001, were also analysed by the PCR-

based inc/rep typing method.

2.2. PCR-based inc/rep typing method

Template DNA was prepared by one of two me-

thods. Method 1 consisted of resuspending a single

colony directly in the 25 Al of PCR reaction mixture;

this method was used during the initial testing of

primers on positive controls but it is not recommended

for the screening of replicons on uncharacterized plas-

mids, because the sensitivity of the PCR using this

template DNA is quite low. Method 2 generated total

DNA of higher purity and followed the Wizard Geno-

mic DNA purification System (Promega, Madison,

WI), starting from 2 ml of LB-broth cultures contai-

ning a suitable concentration of antibiotic for selection;

this method was used in the inc/rep typing of reference

plasmids and on the collection of Salmonella strains,

using no more than 200 ng total DNA per reaction.

Specific primer pairs were designed for eighteen

different basic replicons (Table 1).

Primers for HI1, HI2, I1, X, L/M, FIA, FIB, W, Y,

P, K and B/O replicons were designed based on

sequences obtained by DNA sequencing of the inserts

cloned in the respective pULB inc/rep constructs,

Table 1

Primers used in this study

Name DNA sequence Target site EMBL accession no. Amplicon size (bp)

HI1 FW 5V-ggagcgatggattacttcagtac-3V parA-parB AF250878 471

HI1 RV 5V-tgccgtttcacctcgtgagta-3VHI2 FW 5V-tttctcctgagtcacctgttaacac-3V iterons BX664015 644

HI2 RV 5V-ggctcactaccgttgtcatcct-3VI1 FW 5V-cgaaagccggacggcagaa-3V RNAI M20413 139

I1 RV 5V-tcgtcgttccgccaagttcgt-3VX FW 5V-aaccttagaggctatttaagttgctgat-3V ori g Y00768 376

X RV 5V-tgagagtcaatttttatctcatgttttagc-3VL/M FW 5V-ggatgaaaactatcagcatctgaag-3V repA,B,C U27345 785

L/M RV 5V-ctgcaggggcgattctttagg-3VN FW 5V-gtctaacgagcttaccgaag-3V repA NC_003292 559

N RV 5V-gtttcaactctgccaagttc-3VFIA FW 5V-ccatgctggttctagagaaggtg-3V iterons J01724 462

FIA RV 5V-gtatatccttactggcttccgcag-3VFIB FW 5V-ggagttctgacacacgattttctg-3V repA M26308 702

FIB RV 5V-ctcccgtcgcttcagggcatt-3VW FW 5V-cctaagaacaacaaagcccccg-3V repA U12441 242

W RV 5V-ggtgcgcggcatagaaccgt-3VY FW 5V-aattcaaacaacactgtgcagcctg-3V repA K02380 765

Y RV 5V-gcgagaatggacgattacaaaacttt-3VP FW 5V-ctatggccctgcaaacgcgccagaaa-3V iterons M20134 534

P RV 5V-tcacgcgccagggcgcagcc-3VFIC FW 5V-gtgaactggcagatgaggaagg-3V repA2 AH003523 262

FIC RV 5V-ttctcctcgtcgccaaactagat-3VA/C FW 5V-gagaaccaaagacaaagacctgga-3V repA X73674 465

A/C RV 5V-acgacaaacctgaattgcctcctt-3VT FW 5V-ttggcctgtttgtgcctaaaccat-3V repA K00053 750

T RV 5V-cgttgattacacttagctttggac-3VFIIS FW 5V-ctgtcgtaagctgatggc-3V repA AE006471 270

FIIS RV 5V-ctctgccacaaacttcagc-3VFrepB FW 5V-tgatcgtttaaggaattttg-3V RNAI/repA AY234375 270

FrepB RV 5V-gaagatcagtcacaccatcc-3VK/B FW 5V-gcggtccggaaagccagaaaac-3V RNAI M93063 160

K RV 5V-tctttcacgagcccgccaaa-3VB/O RV 5V-tctgcgttccgccaagttcga-3V RNAI M28718 159

A. Carattoli et al. / Journal of Microbiological Methods 63 (2005) 219–228 221

kindly provided to us by Werner K. Maas (Couturier

et al., 1988). DNA sequences were determined by

fluorescent dye-labeled dideoxynucleotides and a

373 automatic DNA sequencer (Perkin-Elmer, Foster

City, CA). Comparative analysis of nucleotide

sequences was performed by the advanced BLAST

search program 2.0 within the QBLAST system at the

National Center for Biotechnology Information site

(www.ncbi.nlm.nih.gov/blast/).

Primers for the N, FIC, A/C, T and F replicons

were designed based on the published sequences of

R46 (GeneBank accession no. NC_003292), FIlac(GeneBank accession no. AH003523), pRA1 (Gene-

Bank accession no. X73674), Rts1 (GeneBank acces-

sion no. K00053) and NR1 plasmids (GeneBank

accession no. X02302), respectively. All the primers

were designed on the basis of the multiple compara-

tive analysis of nucleotide sequences on the EMBL

Gene Databank. The primer pairs recognize the cod-

ing sequence of the repA genes, the cis-repeats of the

origin of replication or the countertranscript RNAI

(Table 1). The primer pair for the HI1 replicon was

designed based on the parA–parB genes, which con-

trol plasmid partitioning (Table 1). Primer pair FIISFW and FIIS RV (Table 1) were previously described

to recognize the FIIA replicon of Salmonella (FIIAS)

virulence plasmids (Guerra et al., 2002). Because of

the high level of homology between the K and B/O

Table 2

Results of the inc/rep PCR-based method on 61 plasmids of known incompatibility groups

Plasmid testeda Origin1 Inc group Multiplex�inc/rep PCR (base pairs) Simplex�inc/rep

PCR (base pairs)

Ref b

1 2 3 4 5

HI1

(471)

HI2

(644)

I1�Ig

(139)

X

(376)

L/M

(785)

N

(559)

FIA

(462)

FIB

(702)

W

(242)

Y

(765)

P

(534)

FIC

(262)

A/C

(465)

T

(750)

FIIAS

(270)

F

(270)

K

(160)

B/O

(159)

R27 S. Typhi HI1 + � � � � � � � � � � � � � � � � � 1

TP154 S. Typhimurium HI1 + � � � � � � � � � � � � � � � � � 2

TP245 HI2 � + � � � � � � � � � � � � � � � � t.s.

R478 S. marcescens HI2 � + � � � � � � � � � � � � � � � � 1

TP167 HI2 � + � � � � � � � � � � � � � � � � t.s.

TP116 HI2 � + � � � � � � � � � � � � � � � � t.s.

R483 E. coli I1 � � + � � � � � � � � � � � � � � � 1

R144 S. Typhimurium I1 � � + � � � � � � � � � � � � � � � 1

JR66a K. pneumoniae I1 � � + � � � � � � � � � � � � � � � 1

T-Ddrp I1 � � + � � � � � � � � � � � � � � � t.s.

R621a S. Typhimurium Ig � � + � � � � � � � � � � � � � � � 1

TP114 E. coli I2 (Iy) � � � � � � � � � � � � � � � � � � 1

R6K X � � � + � � � � � � � � � � � � � � 1

pIP135 Enterobacter spp. L/M � � � � + � � � � � � � � � � � � � 1

pSem S. Typhimurium L/M � � � � + � � � � � � � � � � � � � 3

R471a S. marcescens L/M � � � � + � � � � � � � � � � � � � 1

R69 Providencia spp. L/M � � � � � � � � � � � � � � � � � � 1

R446b P. morganii L/M � � � � � � � � � � � � � � � � � � 1

33R404 M � � � � + � � � � � � � � � � � � � t.s.

35R318 M � � � � + � � � � � � � � � � � � � t.s.

N3 Shigella spp. N � � � � � + � � � � � � � � � � � � 1

R46 S. Typhimurium N � � � � � + � � � � � � � � � � � � 1

TP209 S. Typhimurium N � � � � � + � � � � � � � � � � � � t.s.

TP118 N, FI � � � � � + + + � � � + � � � + � � t.s.

FV lac E. coli 31R892 FI � � � � � � + + � � � + � � � + � � t.s.

FV lac E. coli CSH28 FI � � � � � � + + � � � + � � � + � � t.s.

R836 FI � � � � � � + + � � � + � � � + � � t.s.

F-T E. coli FI � � � � � � + + � � � � � � � � � � 2

R162 FI � � � � � � + + � � � � � � � + � � 1

TP243 FIme � � � � � � + + � � � � � � � + � � t.s.

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366D2 S. Typhimurium FIme � � � � � � + + � � � � � � � + � � 4

NTP101 S. Typhimurium FIme � � � � � � + + � � � � � � � � � � 4

TP160 S. Typhi FIme � � � � � � + + � � � � � � � + � � 2

TP181 FIme � � � � � � + + � � � � � � � + � � 4

pSa S. flexneri W � � � � � � � � + � � � � � � � � � 5

NR1 S. Paratyphi B FII � � � � � � � � � � � � � � � + � � 1

O157 E. coli O157:H7 FII � � � � � � � � � � � � � � � + � � t.s.

T-D FII, I1 � � + � � � � � � � � � � � + � � � t.s.

S. 82/10 S. Enteritidis FII, I1 � � + � � � � � � � � � � � + � � � t.s.

S. 115/17 S. Enteritidis FII, I1 � � + � � � � � � � � + � � + � � � t.s.

R1-16 S. Paratyphi B FII � � � � � � � � � � � � � � � + � � 1

R1drd19 S. Paratyphi B FII � � � � � � � � � � � � � � � + � � 1

R136 S. Typhimurium FII � � � � � � � � � � � � � � � + � � 1

ColB-K98 E. coli FIII � � � � � � � � � � � � � � � + � � 6

R124 drd-2 FIV � � � � � � � + � � � � � � � + � � 1

R124 S. Typhimurium FIV � � � � � � � + � � � � � � � + � � 1

Rbe9 E. coli FIV � � � � � � + + � � � + � � � + � � t.s.

212 FVI � � � � � � � � � � � � � � � + � � t.s.

Folac S. Typhi FV/FO � � � � � � � � � � � � � � � � � � 1

P1-FV lac Y�FI � � � � � � + + � + � + � � � + � � 1, t.s.

RP4 P. aeruginosa P � � � � � � � � � � + � � � � � � � 1

pIP40a P. aeruginosa A/C � � � � � � � � � � � � + � � � � � 1

33R645 C � � � � � � � � � � � � + � � � � � t.s.

Rts1 P. vulgaris T � � � � � � � � � � � � � + � � � � 1

R391 P. rettgeri J � � � � � � � � � � � � � � � � � � 1

39R54 J � � � � � � � � � � � � � � � � � � t.s.

39R60 K � � � � � � � � � � � � � � � � + + t.s.

R387 S. flexneri K � � � � � � � � � � � � � � � � + + 1

RHH72 B � � � � � � � � � � � � � � � � � + t.s.

TP113 S. Typhimurium B � � � � � � � � � � � � � � � � � + 7

R16 E. coli B/O � � � � � � � � � � � � � � � � � + 1

a Plasmids were from the collection of the Istituto Superiore di Sanita, Rome, Italy and from the collection of the Health Protection gency, Colindale, United Kingdom. Blank lines

in the origin column indicate that the bacterial species originally carrying the plasmids is unknown.b t.s.: this study; plasmids with Inc group confirmed by hybridization with replicon probes (Couturier et al., 1988) or by conjuga n with reference plasmids (Datta and Hedges,

1971); 1—Couturier et al., 1988; 2—Anderson et al., 1977; 3—Villa et al., 2000; 4—Carattoli et al., 2001; 5—Hedges and Datta, 197 ; 6—Frydman and Meynell, 1969; 7—Grindley

et al., 1972.

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A

tio

1

A. Carattoli et al. / Journal of Microbiological Methods 63 (2005) 219–228224

replicons the same forward primer was used in both

these simplex PCRs (Table 1).

The PCR-based inc/rep typing method consists of

five different multiplex-PCRs recognizing three dif-

ferent replicon types, and three simplex-PCRs for F, K

and B/O (Table 2). All PCR amplifications, except the

F-simplex, were performed with the following ampli-

fication scheme: 1 cycle of denaturation at 94 8C for 5

min, followed by 30 cycles of denaturation at 94 8Cfor 1 min, annealing at 60 8C for 30 s and elongation

at 72 8C for 1 min. The amplification was concluded

with an extension program of 1 cycle at 72 8C for 5

min. The F-simplex PCR was performed with the

same amplification program but at an annealing tem-

perature of 52 8C.

2.3. Positive controls

Plasmids pULB-2436, -2433, -2428, -2405, -2423,

-2154, -2404, -2426, -2410, -2420, -2439, and -2406

from the inc/rep plasmid bank were initially used as

positive controls for the respective replicons (Coutur-

ier et al., 1988). The R46, FlacV from E. coli CSH28,

pIP40a, Rts1, S. 82/10 and NR1 plasmids were used

as positive controls to test the N, FIC, A/C, T, FIIAS

and F primer pairs, respectively. All the amplicons

obtained were cloned into a TA-cloning vector (Invi-

trogen-Life Technologies, Milano, Italy) and

sequenced. The cloned amplicons were then used as

positive controls for the multiplex and simplex PCRs.

3. Results and discussion

3.1. Specificity of the PCR-based inc/rep typing

method on reference plasmids

Eighteen specific primer pairs were designed, on

the basis of the multiple comparative analysis of

nucleotide sequence on the EMBL Gene Databank,

for HI1, HI2, I1-Ig, X, L/M, N, FIA, FIB, W, Y, P,

FIC, A/C, T, FIIAS, F, K and B/O replicons. The

specificity of each primer pair, listed in Table 1, was

initially performed on bacterial genomic DNA

extracted from the plasmid-free E. coli DH5a strain

(Invitrogen-Life Technologies). None of the primer

pairs gave positive amplification results on this tem-

plate. For each PCR amplification positive controls

were cloned and sequenced as described in Materials

and methods.

Fifty-eight plasmids belonging to 20 Inc groups

(Table 2), whose replicons are recognized by the set

of primers listed in Table 1, were chosen among our

plasmid collections. Two IncJ plasmids and one IncI2

(Iy) plasmid were also included as negative controls;

since there were no DNA sequence information avail-

able for these replicons they were not included in the

method.

The specificity of the PCR-based inc/rep typing

was then tested on 61 reference plasmids (58 positives

and 3 negatives) originally isolated from 16 different

bacterial species (Table 2).

3.1.1. Multiplex 1

This reaction was designed to recognize HI1-, HI2-,

I1/Ig-carrying plasmids, producing 471 bp, 644 bp

and 139 bp amplicons, respectively. Results obtained

on the 61 plasmids revealed that all the IncHI1,

IncHI2 and IncI1/Ig (also called Ia) plasmids tested

were correctly recognized by Multiplex 1. No false

positive results were observed on the collection of

plasmids. Several IncHI2 plasmids produced the

expected amplicon plus higher molecular mass ampli-

cons (data not shown). These extra bands may be

explained by the presence of multiple iterons located

upstream of the HI2 replicon that can be recognized

by the HI2 RV primer (Gilmour et al., 2004). This

reaction is very specific and positive PCR results,

even in presence of multiple amplicons, should be

interpreted as positive for the presence of the HI2

replicon.

The IncI2 (Iy) plasmid included in the collection

was negative in all the PCR amplifications. This was

as expected because this plasmid is classified into a

different subgroup in respect to the IncI1/Ig plasmids

and also exhibits a different pilus serotype (Couturier

et al., 1988).

3.1.2. Multiplex 2

This reaction was designed to recognize the X, L/

M and N replicons by amplification of 376 bp, 785 bp

and 559 bp amplicons, respectively. When used on the

61 reference plasmids, this reaction correctly detected

all the IncX and IncN plasmids and gave no false

positive results with other replicons. Multiplex 2

failed in detecting two of the seven plasmids belong-

A. Carattoli et al. / Journal of Microbiological Methods 63 (2005) 219–228 225

ing to the IncL/M group (R69 and R446b in Table 2).

R69 and R446b were positively hybridized by South-

ern blot hybridization using the L/M cloned amplicon

as a probe (data not shown). These results suggest that

the L/M replicons may diverge in sequence and occa-

sionally give false negative results by PCR. Since

only a few DNA sequences are available for these

replicons, the higher sensitivity hybridization-based

method can be applied to recognize them.

3.1.3. Multiplex 3

This reaction recognizes the FIA, FIB and W

replicons, producing 462 bp, 702 bp and 242 bp

amplicons, respectively. This amplification success-

fully recognized all the IncFI plasmids, including

IncFIme plasmids (Anderson et al., 1977), as well as

the IncW plasmid pSa (Okumura and Kado, 1992),

producing the respective amplicon (Table 2). The

IncFIV R124drd-2, R124 and Rbe9 plasmids were

positive for the FIB replicon (Table 2), and Rbe9

plasmid was also positive for FIA and FIC. These

results reproduce previously described replicon typing

results obtained by inc/rep hybridization. (Couturier et

al., 1988; Campbell and Mee, 1989).

3.1.4. Multiplex 4

This reaction was designed to recognize IncY and

IncP plasmids and also included a primer pair spe-

cific for the FIC replicon (262 bp) of the E. coli FVlac fertility plasmid. The IncY P1 and the IncP RP4

(Smith and Thomas, 1987) plasmids were positive

for Y (765 bp) and P (534 bp) replicons, respec-

tively. The FIC PCR was designed to recognize the

repA2 gene located on the E. coli FV lac plasmid and

does not recognize the FIC of the IncFIme plasmids.

This is recognized by the simplex F-PCR as dis-

cussed below.

The IncY P1 plasmid was also unexpectedly posi-

tive for FIA, FIB and FIC (Table 2). Further charac-

terization of this plasmid revealed the presence of a

multireplicon fusion of the plasmid P1 with the FV lacplasmid (data not shown), confirming the specificity

of the inc/rep PCR-based method in detecting both FI

and Y replicons.

3.1.5. Multiplex 5

This reaction was designed to recognize the IncA/

C, IncT plasmids and the repFIIA replicon of the

virulence plasmids of S. enterica serovars Enteriditis,

Dublin, Cholerae-suis and Typhimurium (Guerra et

al., 2002; Llanes et al., 1996), producing 465 bp,

750 bp and 270 bp PCR products, respectively. The

reference plasmids of the A/C and T groups were

correctly identified by the PCR-based method. Three

S. enterica serovar Enteritidis strains (82/10, 115/17

and T-D) carrying both the virulence and the IncI1

plasmids, were positively recognized by the FIIs and

I1/Ig reactions as expected. This experiment demon-

strated that multiple plasmids simultaneously present

in the same cell can be easily detected by this PCR-

based method.

3.2. The three simplex-PCRs for the F, K and B/O

replicons

Particular attention has been paid to the design of

primer pairs recognizing the I1, K and B/O repli-

cons. The I1, K and B/O replicons were not discri-

minated by the hybridization-based inc/rep typing,

sharing a DNA sequence homology higher than

90% (Couturier et al., 1988). The incompatibility

phenotype of this class of plasmids is an expression

of the evolutionary changes in the DNA sequence of

the RNA inhibitor (RNAI). Few base substitutions

within the major stem-and-loop structures predicted

for analogous RNAI of different plasmids lead to

compatible phenotypes. The specificity of our assay

relies on the specific I1/Ig PCR in Multiplex 1 and

in two simplex PCRs designed to detect the K and

B/O replicons. In the conditions used, the B/O-sim-

plex specifically detects the B/O replicon while the

K-simplex recognizes both the K and B/O (Table 2),

allowing the identification and discrimination of both

replicons.

The F amplification reaction was designed to

recognize, by a single PCR, all the plasmids belong-

ing to the heterogeneous group of the IncF-plasmids.

This reaction was based on the highly conserved

region of homology showed by the FIC and FII

replicons (Saadi et al., 1987). This PCR amplifica-

tion recognized 20 of the 26 FI, FII, FIII, FIV, FV

and FVI plasmids tested, including the FII replicon

of the E. coli O157 virulence plasmid. Among the

six F-plasmids not recognized by the F-simplex,

three were Salmonella virulence plasmids, whose

FII replicon was specifically recognized in the Multi-

A. Carattoli et al. / Journal of Microbiological Methods 63 (2005) 219–228226

plex 5 reaction and three were IncFIme plasmids that

probably lack the FIC replicon but were positive for

the FIA and FIB replicons by Multiplex 3 (Couturier

et al., 1988). The Folac plasmid assigned to the

IncFV/FO group was also negative for the F-sim-

plex. This plasmid was previously reported to give

negative results by hybridization with the inc/rep

probes (Couturier et al., 1988), and its replication

controls have never been analyzed at a DNA sequence

level.

In conclusion, the PCR-based method demon-

strates high specificity and sensitivity in detecting

replicons on reference plasmids. This method is

more specific than the hybridization-based method.

This was particularly evident when applied to FII,

FIC, I1-Ig, K and B/O replicons that exhibit consider-

able cross-reactions by hybridization-based inc/rep

typing, thus limiting the use of this method in the

classification of plasmids (Couturier et al., 1988).

Although these eighteen replicons are representative

of the major plasmid incompatibility groups among

the family Enterobacteriaceae, several other replicons

Table 3

PCR-based replicon typing applied to a collection of multidrug resistant S

Isolate Source Serotype Resistancea

17/24 animal Agona SmSxTTe

17/20 bovine meat Anatum GmNaSmSxTTe

17/17 turkey Blockley CfKmNaNmSmTe

17/34 meat food Blockley KmNaNmSmTe

17/41 swine meat Blockley KmNmSmTe

17/8 turkey Bredeney ApAmcCfCmEnNaS

17/3 dog stool Bredeney KmNmSmSuTe

27/30 pork sausage Give ApSmSxTTe

17/16 duck Hadar ApAmcCfKmNmSm

17/9 duck Hadar ApNmSmSxTTe

17/37 chicken Hadar ApCfEnNaSmTe

17/35 chicken Hadar ApAmcCfEnNaSmT

17/4 turkey Heidelberg ApAmcNaSmTe

17/29 swine meat Heidelberg ApCfSmSxTTe

17/6 swine Heidelberg CmGmKmNmSmSx

17/40 chicken London ApAmcCfEnNaSuS

17/31 swine London SmSuTe

17/27 turkey Saintpaul ApAmcCfGmKmNa

17/21 turkey Saintpaul ApAmcCfEnGmKm

17/13 chicken Senftenberg ApAmcCfClCtxSmS

a Ampicillin (Ap), amoxicillin�clavulanic acid (Amc), cephalothin (Cf)

acin (En), streptomycin (Sm), sulfonamides (Su), tetracycline (Te), trimeth

nalidixic acid (Na), neomycin (Nm), cefotaxime (Ctx). Strains showinb strA–strB, tet(A) and the integrons (int) were previously described (P

should be cloned and sequenced to provide a complete

typing scheme of all known plasmids and to extend

the application of this method to a wider range of

bacterial species.

3.3. Application of the PCR-based inc/rep typing

method to a collection of multidrug resistant S. enter-

ica isolates

The PCR method was then applied to a collection

of 20 epidemiologically-unrelated multidrug-resistant

S. enterica strains of 10 different serotypes isolated in

Italy (Table 3). These isolates belong to a well char-

acterized collection, previously analyzed for antimi-

crobial resistance genes (Pezzella et al., 2004). Awide

diffusion of the strA-strB and tet(A) resistance genes

was observed among the strains and 7 of 20 strains

also carried integrons encoding resistance to trimetho-

prim, kanamycin, sulfonamides and streptomycin

(Pezzella et al., 2004).

Table 3 shows the results obtained from these

strains by the PCR-based inc/rep typing method.

. enterica isolates

inc/rep strAb tetAb intb

HI2 + + +

HI2 + + +

N + + �N, A/C + + �N + + �

mSxTTe HI2, I1 + + +

neg + � �I1 + + +

Te X + + �X + + �N + + �

e neg + + �N + + �N � + �

TTe A/C � + +

mTe A/C + + �neg + + �

NmSmSuTe HI2 + � +

NaNmSmSxTTe HI2 + � +

xT neg + � �, colistin (Cl), chloramphenicol (Cm), ciprofloxacin (Cp), enroflox-

oprim-sulfamethoxazole (SxT), kanamycin (Km), gentamicin (Gm),

g both Su and SxT resistance phenotypes are reported as SxT.

ezzella et al., 2004).

A. Carattoli et al. / Journal of Microbiological Methods 63 (2005) 219–228 227

The inc/rep typing detected the presence of N, I1,

HI2, A/C and X replicons in several isolates of this

collection. Four strains were negative for all the

replicons tested. The N and the HI2 replicons were

detected in six and four strains, respectively, belong-

ing to different serotypes (Agona, Anatum, Blockley,

Bredeney, Hadar, Heidelberg and Saintpaul). Three

strains were positive for the A/C replicon and the X

replicon was identified in two Hadar isolates. Inte-

grons were associated with I1-, HI2-and A/C-positive

strains, but not with the N-positive strains (Table 3).

These results suggest the presence of recurrent and

common plasmids in epidemiological unrelated Sal-

monella isolates of different serotypes isolated from

distant geographical areas in Italy, suggesting the

successful spread of these genetic determinants in

zoonotic pathogens.

This analysis demonstrates that the PCR-based

method can be applied directly on collections of

strains, suggesting the potential use of the PCR-

based replicon typing method in tracking the spread

of plasmids conferring drug resistance. The method

could be applied to monitor the circulation of plas-

mids within strains from different environments or to

follow the horizontal transmission of antimicrobial

resistance genes among the Enterobacteriaceae.

Acknowledgments

We are grateful to V. Miriagou, L.S. Tzouvelekis,

E. Tzelepi and G.M. Rossolini for helpful discussion

and continuous encouragement. We thank Fabio Ric-

cobono for the DNA sequencing. This work was

supported by grants from the Istituto Superiore di

Sanita (Art. 502, project no. 2012/RI; Art. 524, project

no. C3MD).

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