Evaluation of a novel method based on amplification of DNA fragments surrounding rare restriction...

Evaluation of a novel method based on amplification of DNA

fragments surrounding rare restriction sites (ADSRRS

fingerprinting) for typing strains of vancomycin-resistant

Enterococcus faecium

Beata Krawczyka, Krzysztof Lewandowskia, Marek Bronkb, Alfred Sametb,Przemyslaw Myjakc, Jozef Kura,*

aDepartment of Microbiology, Technical University of Gdansk, ul. G. Narutowicza 11/12, 80-952 Gdansk, PolandbDepartment of Clinical Bacteriology, State Hospital No. 1, ul. Debinki 7, 80-211 Gdansk, Poland

c Institute of Maritime and Tropical Medicine, 81-519 Gdynia, ul. Powstania Styczniowego 9 B, Poland

Received 17 April 2002; received in revised form 23 July 2002; accepted 6 September 2002

Abstract

In the search for an effective DNA-typing technique for use in hospital epidemiology, the performance and convenience of a

novel assay based on the fingerprinting of bacterial genomes by amplification of DNA fragments surrounding rare restriction

sites (ADSRRS fingerprinting) was tested. A large number of vancomycin-resistant Enterococcus faecium (VREM) isolates

from haematological ward patients of the Clinical Hospital in Gdansk were examined. We found that ADSRRS fingerprinting

analysis is a rapid method that offers good discriminatory power. The method demonstrated also excellent reproducibility. The

usefulness of the ADSRRS fingerprinting method for molecular typing was compared with pulsed field gel electrophoresis

(PFGE) method, which is currently considered the gold standard for molecular typing of isolates recovered from patients and

the environment in the course of investigation and control of nosocomial outbreaks. Clustering of ADSRRS fingerprinting data

matched pulsed field gel electrophoresis data.

The features of ADSRRS fingerprinting technique is discussed in comparison with conventional methods. Data presented

here demonstrate the complexity of the epidemiological situation concerning VREM that may occur in a single medical ward.

D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Enterococcus faecium; PCR; PCR fingerprinting; PCR suppression; Vancomycin-resistant enterococci

1. Introduction

Over the past 10 years, a number of Enterococcus

strains with high-level inducible resistance to vanco-

mycin have been identified, and the relative incidence

of these strains has increased sharply in the last years.

In addition, many reports of nosocomial outbreaks of

infection with VRE have been published, especially

from North America and Europe (Karanfil et al.,

1992; Handwerger et al., 1993; Jordens et al., 1994;

Montecalvo et al., 1994). Recently, the isolation of a

vancomycin-resistant Enterococcus faecium (VREM)

in Poland was reported (Samet et al., 1999; Hrynie-

0167-7012/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

PII: S0167 -7012 (02 )00187 -2

* Corresponding author. Tel./fax: +48-58-3471822.

E-mail address: [email protected] (J. Kur).

www.elsevier.com/locate/jmicmeth

Journal of Microbiological Methods 52 (2003) 341–351

wicz et al., 1999; Kawalec et al., 2000). The E.

faecium PCR-based specific diagnostic assay was

used for confirmation of the phenotype identification

of E. faecium (Cheng et al., 1997) and the multiplex

PCR-restriction fragment length polymorphism me-

thod for type of Van resistance (Patel et al., 1997). To

determine whether the isolates were epidemiologically

related, isolated strains were differentiated by PCR

fingerprinting method (Samet et al., 1999). The

applied PCR fingerprinting system allowed for the

differentiation of the clinical isolates from the Hae-

matological Unit. Two main strains (genotypes) were

identified. The PCR fingerprinting of VREM from the

Haematological Unit demonstrated only small genetic

heterogeneity among the isolates over 11 months, with

two strains being identified. These strains were genet-

ically closely related. In the present study, 100 VREM

strains (including 25 strains described previously;

Samet et al., 1999) within a duration of 36 months

(between January 1997 and December 1999) taken

from 100 patients were examined using a novel

fingerprinting method (ADSRRS fingerprinting)

described by Masny and Plucienniczak (2001). The

utility of the ADSRRS fingerprinting method was

evaluated with data obtained using pulsed field gel

electrophoresis (PFGE) method.

Genomic fingerprints are increasingly used to

study relationships at the intra- or even interspecific

level. The fingerprints are obtained by visualising

many parts of the genome. Differences in these finger-

prints between individuals are interpreted as genetic

distances. Obviously, the differences should reflect

variations in DNA rather than artifacts due to a non-

robust method. Furthermore, the method should pro-

vide the appropriate level of discriminatory power and

it should be relatively rapid and cheap, especially in

large-scale population genetic studies.

Macrorestriction analysis of genomic DNA fol-

lowed by pulsed field gel electrophoresis (PFGE)

has become the ‘‘gold standard’’ for molecular typing.

However, PFGE is limited in its resolving power

(Gerner-Smidt et al., 1998), and this contributes to

difficulties with gel-to-gel and interlaboratory repro-

ducibility (Van Belkum et al., 1998).

A variety of PCR-based methods for displaying

DNA sequence polymorphism have been developed.

Some of the methods such as RAPD (Williams et al.,

1990) and AFLP (Vos et al., 1995) do not require prior

knowledge of the DNA sequence. RAPD is generally

regarded to be less time consuming, while the AFLP

method is more robust (Mueller and Wolfenbarger,

1999; Pejic et al., 1998). RAPD allows detection of

DNA polymorphisms between strains of a species but

does not exhibit high rates of reproducibility. The

performance of RAPD is sensitive to many factors

such as selection of primers, magnesium concentra-

tion in the PCR buffers and the thermocycler used for

PCR (De Zoysa and Efstratiou, 1999). An amplified

restriction fragment polymorphism (AFLP) over-

comes many of the problems of RAPD. There are

three major steps in the AFLP procedure: (i) restric-

tion endonuclease digestion of genomic DNA and the

ligation of specific adapters; (ii) amplification of the

restriction fragments by PCR using primer pairs con-

taining common sequences of the adapter and one to

three arbitrary nucleotides; (iii) analysis of the ampli-

fied fragments using gel electrophoresis. The combi-

nation of different restriction enzymes and the choice

of selective nucleotides in the primers for PCR make

AFLP a useful system for molecular typing of micro-

organisms but requires the use of sequencing gels and

usually labeled primers because of the quantity of

simultaneously amplified DNA fragments.

Here, we show evaluation of a novel fingerprinting

method described by Masny and Plucienniczak (2001)

based on the fingerprinting of bacterial genomes by

amplification of DNA fragments surrounding rare

restriction sites (ADSRRS fingerprinting) for epide-

miological studies. This method is based on the

digestion of total bacterial DNA with two restriction

enzymes differing in cleavage frequency, ligation with

two different oligonucleotide adapters and suppres-

sion of PCR (Lukyanov et al., 1994; Diatchenko et al.,

1996; Shagin et al., 1999). PCR suppression allows

the amplification of only a limited subset of DNA

fragments, as only those with two different oligonu-

cleotides ligated at the ends of complementary DNA

strands are amplified in the PCR. The method does

not require prior knowledge of the sequence of the

analyzed DNA and generates a limited number of

DNA fragments, whose band pattern on the gel differs

between strains of a bacterial species. Furthermore,

the DNA fragments can be easily analyzed on poly-

acrylamide gels stained with ethidium bromide. We

have implemented this method using a set of clinical

vancomycin-resistant E. faecium (VREM) isolated

B. Krawczyk et al. / Journal of Microbiological Methods 52 (2003) 341–351342

from Haematological Unit patients of the Clinical

Hospital in Gdansk.

2. Materials and methods

2.1. Isolates and patients

The first VREM isolate was cultured in the Uni-

versity Hospital in Gdansk from the urinary tract

infection of a patient located in the Haematological

Unit in December 1996. From January 1997 to Decem-

ber 1999, about 15,000 clinical samples were exam-

ined for the presence of E. faecium isolates and the

isolates were tested for the resistance to vancomycin

and teicoplanin. A total of 587 E. faecium isolates were

recovered and 344 of them were vancomycin- and

teicoplanin-resistant. A hundred isolates were chosen

for further examination by molecular typing methods

(one isolate from one patient). VREM were isolated

from blood (22), urine (13), stool (58), sputum (2), pus

(2), skin (1), vagina (1) and throat (1). Some clinical

data of patients colonised or infected with vancomy-

cin-resistant E. faecium are presented in Table 1.

2.2. DNA isolation

DNA isolations (from 1.5 ml of culture) were

carried out with the Genomic DNA Prep Plus (A&A

Biotechnology, Poland) according to the manufactures

procedure with minor modifications. For disruption of

enteroccocal cells, the incubation at 37 jC for 30 min,

before DNA isolation, with 2.5 mg of lysosyme

(Sigma-Aldrich Chemie, Steinheim, Germany) and

100 Ag lysostaphin (Sigma, St. Louis, MO, USA)

per 1 ml of TE buffer was applied. DNA quantities in

the samples were estimated by electrophoresis of 1 AlDNA solution on 1% agarose (Sigma) gels run

together with samples with known amounts of DNA

and subsequent ethidium bromide staining (Sigma-

Aldrich) in 0.5 mg/l solution for 10–15 min. The

DNA concentration range was from about 100 ng/Alto several hundred nanograms per microliter.

2.3. PCR

A PCR assay for identification of E. faecium and

primers used were the same as in Cheng et al. (1997),

with some modifications as described by Samet et al.

(1999). A multiplex PCR-restriction fragment length

polymorphism (MPCR/RFLP) assay for Van-type

identification of E. faecium isolates were carried out

according to Patel et al. (1997). Amplification prod-

ucts obtained with Van-specific primers were further

analysed by digestion with MspI restriction endonu-

clease (RFLP).

2.4. ADSRRS fingerprinting

Enterococcal DNA (100–500 ng) was digested

with a combination of two enzymes: XbaI (10 U/Al)(Sigma) and BglII (10 U/Al) (Sigma) for 2–3 h at 37

Table 1

Selected clinical details of patients infected or colonised with vancomycin-resistant E. faecium

Year of Age/sex/ Diagnosisa/ Source/no. of isolates

isolation no. of patient no. of patientInfection Colonisation

1997 19–54/F/14, 16–61/M/19 AML/14, CML/14,

ALL/4, NHL/1

blood/9, urine/2 stool/20, sputum/1, throat/1

1998 20–59/F/14, 20–77/M/18 AML/14, CML/7,

ALL/6, NHL/2, AA/3

blood/6, urine/6 stool/18, vagina/1, skin/1

1999 9–77/F/13, 4–52/M/22 AML/11, CML/12,

ALL/3, NHL/3, AA/1,

CABG/3, UTI/1, TR/1

blood/7, urine/5 stool/20, sputum/1, pus/2

1997–1999 9–77/F/41, 4–77/M/59 AML/39, CML/33, ALL/13,

NHL/6, AA/4, CABG/3,

UTI/1, TR/1

blood/22, urine/13,

pus/2

stool/58, sputum/2, throat/1,

vagina/1, skin/1

Total 100

a AML, acute myeloid leukaemia; CML, chronic myeloid leukaemia; ALL, acute lymphoid leukaemia; NHL, non-Hodgkin’s lymphoma;

AA, aplastic anaemia; CABG, coronary artery bypass graft; UTI, urinary tract infection; TR, trauma.

B. Krawczyk et al. / Journal of Microbiological Methods 52 (2003) 341–351 343

jC in a total volume 50 Al of appropriate buffer. To

each of the digested samples, 20 Al 3 M NaAc, 150 AlTE buffer, 400 Al 96% ethanol and 3 Al glycogen (20

mg/ml) (Sigma) were added. Samples were vortexed,

mixed and incubated at � 20 jC for 60 min, then

centrifuged at 12,000� g for 10 min. Ethanol was

removed and the pellets were washed with 200 Al of70% ethanol, then centrifuged for 5 min at 12,000� g

and again ethanol was removed, and pellets were

dried for 20 min at room temperature.

Adapters were assembled from two oligonucleoti-

des (Table 2). Oligonucleotides were dissolved in

water to concentration of 20 pmol/Al (each oligonu-

cleotide), then heated at 90 jC in a water bath for 2 min

and subsequently left at room temperature for 10 min to

anneal. Appropriate adapters were ligated to the cor-

responding cohesive ends (Table 2 and Fig. 1). Dry

pellets of DNA digests were dissolved in a solution

consisting of 2 Al 10� ligation buffer (66 mM Tris–

HCl, pH 8.5, 6.6 mM MgCl2, 10 mM DTT, 66 AMATP), 1 Al (20 pmol each adapter) of the solution of the

adapters corresponding to the cohesive ends left by the

enzymes used for the prior digestion of the sample, 0.5

Al (1 U/Al) T4 DNA ligase (Epicentre, USA), 2 Al 50%PEG 4000 solution and water to 20 Al. The ligation

reactions were carried out at 16 jC for 2 h. After

ligation, 80 Al of TE buffer was added, and DNAwas

isopropanol precipitated according to standard proce-

dure. After centrifugation at 12,000� g for 10 min, the

DNA pellets were dissolved in 20 Al TE buffer.

The PCR reaction was carried out in a 50-Alreaction mixture containing 2 Al ligation solution, 5

Al PCR buffer (100 mM Tris–HCl, pH 8.8, 500 mM

KCl, 20 mM MgCl2, 1% Triton X-100), 5 Al of a

deoxynucleoside triphosphate (dNTP) mixture (con-

centration of each dNTP, 2.5 mM), 10 Al betaine (5 M

solution; Sigma), 1 Al (1U) of Taq polymerase

(Shark2, DNA Gdansk II, Poland) or 1 Al (1U) of

Pwo polymerase (DNA Gdansk II), 50 pM each RC

and FC primer (Table 2) and water to 50 Al. Thermal

cycl ing was performed in a Perkin-Elmer

GENEAMPR PCR System 2400 or Hot-Shot24 ther-

mal cycler (DNA Gdansk II). The following thermal

profile was applied: an initial cycle at 94 jC for 5 min

and then at 72 jC for 5 min for filling the ends of the

DNA fragments, followed by denaturation at 94 jCfor 5 min, then 19 cycles of 94 jC for 30 s, 62 jC for

30 s, 72 jC for 90 s, followed by 5 min at 72 jC. PCRproducts (10 of 50 Al) were electrophoresed on 6%

polyacrylamide gels with TBE buffer, stained in

ethidium bromide at 0.5 mg/l aqueous solution for

10–15 min and images of the gels were documented

by photographing using a White/Ultraviolet Trans-

illuminator. The patterns obtained from the electro-

pherograms were converted and analyzed using the

Quanty One software, version 4.3.1 (Bio-Rad, USA).

A UPGMA dendrogram was generated using Dice

correlation coefficient in the Quanty One software.

The reproducibility of the technique was examined by

performing three ADSRRS fingerprinting runs for

each isolate with three separate DNA extractions

and by using two different thermal cyclers (Perkin-

Elmer GENEAMPR PCR System 2400 or Hot-

Shot24 thermal cycler, DNA Gdansk II).

2.5. PFGE

PFGE was performed using Bio-Rad’s GenePath

Group 1 kits. A single colony from a 24-h isolate was

grown overnight in LB under aerobic conditions at 37

jC. Cells were collected and resuspended in cell

suspension buffer. The suspension was mixed with

Table 2

Adapters and PCR primers used in this study

Adaptersa and primers Nucleotides sequences

XbaI short adapter 5V-CTAGGTCGACGTT-3V3V-CAGCTGCAACCACCTACTTCC-5V

oligo-Xba helper,

oligo-Xba-lig

BglII long adapter 5V-GATCCGTCGACAACGGCGTTCCTTCGTCTACCATCC-3V3V-GCAGCTGTTGCCGCAAGGAAGCAGATGGTAGG-5V

oligo-Bgl helper,

oligo-Bgl-lig

XbaI short primer

(oligo-Xba-lig)

5V-CCTTCATCCACCAACGTCGAC-3V

BglII long primer 5V-GGATGGTAGACGAAGGAACGC-3Va Adapters are double stranded but only their constant parts (italic) are ligated, helper parts serve just to create dsDNA fragment and

protruding 5Vend.


Fig. 1. Diagram illustrating the ADSRRS fingerprinting technique used in the study. The blacks circles and triangles mark the point where

ligation occurs.


lysozyme–lysostaphin and embedding agarose, and

plugs were made. Plugs were placed in lyses buffer

with additional lysozyme–lysostaphin and incubated

for 3 h at 37 jC without agitation. The plugs were

washed, placed in proteinase K solution and incubated

overnight at 50 jC without agitation. Plugs were

washed four times with wash buffer for 30–60 min at

room temperature on a rocker.

DNA was digested overnight with 25 U of XbaI

(Sigma) at 37 jC. DNAwas separated on an agarose gel

using the GenePath instrument (based on the pulsed

field method of contoured clamped homogenous elec-

tric field). The run time was 20 h. Gels were stained

with ethidium bromide. The patterns obtained from the

electropherograms were converted and analyzed using

the Quanty One software, version 4.3.1 (Bio-Rad).

3. Results

3.1. PCR identification of E. faecium and Van-type

To confirm the phenotypic identification, the PCR

identification of E. faecium and Van-type of antibiotic

resistance was carried out. Using the EM1A and EM1B

primers (Cheng et al., 1997; Samet et al., 1999), a

specific 658-bp DNA product, upon PCR amplification

of DNA from all isolates identified as E. faecium by

standard biochemical assays, was identified (results not

shown). No amplification was observed with isolates

identified as E. faecalis or E. gallinarum. These results

confirmed that examined isolates in fact belong to E.

faecium. Next, the convenient multiplex PCR-restric-

tion fragment length polymorphism (MPCR/RFLP)

assay to detect and discriminate vanA, vanB and

vanC-1 genes according to Patel et al. (1997) was

applied. All examined clinical isolates, phenotypically

identified as vancomycin-resistant VanA-type of the E.

faecium, yielded the 885-bp amplicon, which is char-

acteristic for vanA and vanB genes. The amplified

DNAs from all isolates digested with MspI restriction

enzyme gave distinct electrophoretic patterns for vanA

gene (231, 184, 163, 133 and 131 bp restriction frag-

ments) (results not shown). These experiments con-

firmed that the isolates belong in fact to VanA-type of

the vancomycin resistance.

3.2. ADSRRS fingerprinting analysis

We applied the newly discovered technique for

fingerprinting of bacterial strains described by Masny

and PlCucienniczak (2001), with some minor modifica-

tions. Their proposal is to amplify exclusively DNA

fragments surrounding relatively rare nucleotide

sequences (e.g., rare restriction sites) and the PCR

suppression (SP PCR) phenomenon is the basis for

obtaining limited representation of the DNA fragments

that form the bacterial genome. The outline of that

method applied in our experiments is shown in Fig. 1.

A genomic total DNA is digested with two restriction

enzymes, rare (XbaI, RC) and frequent (BglII, FC)

cutters. Three kinds of DNA fragments—abundant,

sporadic and limited arise that are formed after diges-

tion with frequent, rare and both cutters at the same

time, respectively. The mixture of DNA fragments is

ligated with two different synthetic adapters (XbaI

short adapter and BglII long adapter). All 5Vends of

the most abundant DNA fragments produced by

digestion with a frequent cutter (FC) are modified by

joining the same synthetic oligonucleotide (oligo-Bgl-

lig). Similarly, sporadic fragments generated by diges-

tion with a rare cutter are modified by ligation of

oligonucleotide oligo-Xba-lig to both 5Vends of eachdsDNA fragment. After filling in of the modifying

oligonucleotides joined to the 5Vends with DNA

polymerase all single-stranded abundant and sporadic

DNA fragments have complementary sequences on

their 5Vand 3Vends and because of that, the proper

usage of suppression PCR (SP PCR) during amplifi-

cation of the genomic fragment mixture should elim-

inate the most and least abundant DNA fragments from

the mixture. However, fragments arising after diges-

Fig. 2. Panel A: ADSRRS fingerprints and UPGMA dendrogram of the vancomycin-resistant E. faecium strains (representative results). The

lane designated M contained molecular mass marker (1008, 883, 615, 517, 466 bp). Lanes 1–10: strains isolated in 1997 at the beginning of

outbreak (from urine—lane 1, from blood—lanes 6 and 9, from sputum—lane 3, from stool—lanes 2, 4, 5, 7, 8 and 10). The remaining lanes

show ADSRRS fingerprints of the sporadic strains representing patterns of the D, E, F, G and H groups. ADSRRS fingerprinting types are given

above each lane. The DNA fragments were electrophoresed in 8% polyacrylamide gel by using 1�Tris–borate EDTA running buffer at a field

strength of 8 V cm� 1. Panel B: PFGE profiles and UPGMA dendrogram of the E. faecium strains (representative results). Numbers refer to

strains shown in panel A. Chromosomal DNA was digested with XbaI, and the fragments were fractionated on a 0.9% agarose gel.


Fig. 2.


Fig. 2 (continued).


tion with rare and frequent restriction enzymes at the

same time and consequently joined with two distinct

modifying oligonucleotides, oligo-Xba-lig oligo-Bgl-

lig, are amplified exponentially. After filling in of the

modifying oligonucleotides joined to the 5V ends with

DNA polymerase, single-stranded fragments do not

have complementary sequences on their 5V and 3Vendsand consequently are not susceptible to SP PCR.

The obtained ADSRRS fingerprinting patterns for

representative isolates are presented on the Fig. 2A.

Each pattern consists of approximately 20–25 frag-

ments in the size range of 50–2000 bp. ADSRRS

fingerprinting patterns of the 100 VREM found eight

unique profiles represented by A to H groups in Table 3

and Fig. 2A. Isolates of VREM recovered from patients

in the Haematological Unit in 1997 were typed into

ADSRRS fingerprinting groups A, B, C, D and E.

Groups A, B, C, D, F, G, H and groups A, B, C, G, H

were detected in 1998 and 1999, respectively. Strains

representing group E disappeared in 1998 and group D

in 1999. Groups F, G and H appeared later in 1998.

Group F is identified only in 1998 and is represented

by two isolates from stool. Two groups, A and B, were

markedly predominant, as these were represented

by 41% and 25%, respectively. Dice coefficients of

pattern similarity of the ADSRRS fingerprints revealed

high similarity of 0.83 only for the isolates of groups

A and B.

It was investigated next whether similar typing

results might be obtained by the use of different

restriction enzymes and oligonucleotide adapters.

ADSRRS fingerprinting analysis with NotI and StyI

enzymes and appropriate adapters exhibited the same

discriminatory power (results not shown).

3.3. ADSRRS fingerprinting validation

The validation of the ADSRRS fingerprinting

method for epidemiological investigation was based

on a set of 10 VREM isolates with comprehensive

epidemiological, microbiological and molecular typ-

ing data from previously described outbreak (Samet et

al., 1999), and eight isolates with no epidemiological

linkage (vancomycin-sensitive E. faecium isolates

from different hospitals). The applied PCR finger-

printing method enabled differentiation of two main

clones genetically closely related (Samet et al., 1999).

Analysis of the ADSRRS fingerprinting data obtained

with those VREM isolates correctly identified the

outbreak isolates and grouped them also into two

groups (results not shown). As expected, the unrelated

epidemiologically isolates show a high degree of

diversity with unique pattern of bands for each strain

(results not shown).

We also validated the reproducibility of the

ADSRRS fingerprinting method. All VREM isolates

were taken from pure culture to ADSRRS fingerprint

on three separate occasions to assess reproducibility

of the method. The resulting ADSRRS fingerprints

from each separate run produced identical profiles.

There was small variation in peak heights but this did

not result in the gain or loss of any information.

Table 3

E. faecium VREM typing by ADSRRS fingerprinting

Year of

isolation

Source/no.

of isolates

ADSRRS fingerprinting groups/

no. of isolates

A B C D E F G H

1997 Infection

blood/9 3 3 1 1 1

urine/2 2

Colonisation

stool/20 12 3 2 1 2

sputum/1 1

throat/1 1

1998 Infection

blood/6 3 2 1

urine/6 3 2 1

Colonisation

stool/18 12 2 1 2 1

vagina/1 1

skin/1 1

1999 Infection

blood/7 3 3 1

urine/5 1 3 1

Colonisation

stool/20 3 6 4 1 6

sputum/1 1

pus/2 2

1997–

1999

Infection

blood/22 9 6 4 1 1 1

urine/13 6 5 2

pus/2 2

Colonisation

stool/58 27 11 7 1 2 2 2 6

sputum/2 1 1

throat/1 1

vagina/1 1

skin/1 1

Total 100 41 25 12 4 4 2 5 7


3.4. PFGE analysis

The molecular typing by PFGE found seven unique

profiles (represented by AB, C, D, E, F, G and H group

in Fig. 2B). Each pattern consists of approximately

12–17 fragments. Isolates of VREM recovered from

patients in 1997 were typed into PFGE groups AB, C,

D and E. The PFGE group AB is identified as groups

A and B by ADSRRS fingerprinting method. Groups

AB, C, D, F, G, H and groups AB, C, G, H were

detected in 1998 and 1999, respectively.

4. Discussion

PFGE, especially when combined with serotyping,

is currently considered the gold standard for molec-

ular typing of isolates recovered from patients and the

environment in the course of investigation and control

of nosocomial outbreaks. However, PFGE is time-

consuming and labor-intensive and can be performed

only in reference laboratories with skillful technicians.

Due to these drawbacks, PFGE is not an ideal typing

method for health departments undertaking routine

analysis of large numbers of isolates.

Here, we show for the first time the evaluation of a

novel fingerprinting method described by Masny and

Plucienniczak (2001) based on the fingerprinting of

bacterial genomes by amplification of DNA fragments

surrounding rare restriction sites (ADSRRS finger-

printing) for epidemiological studies. The high differ-

entiation power of the ADSRRS fingerprinting method

is shown on clinical strains of E. faecium. Fig. 2A

shows a high degree of diversity of the analyzed strains

representing each of eight identified groups (only

groups A and B are closely related). Identical results

were obtained in independent experiments performed

with DNA isolated from different cultures of one strain.

Results obtained in PCRs do not depend on the thermo-

stable DNA polymerase used (Taq polymerase or Pwo

polymerase) or on the thermal cycler used. The use of

betaine improves the PCR efficiency, especially for

high-molecular-weight DNA fragments. There are

several advantages of the ADSRRS fingerprinting

method: (i) the method does not require prior knowl-

edge of an analyzed sequence; (ii) results can be easily

analyzed even on polyacrylamide gels stained with

ethidium bromide; (iii) one set of adapters and enzymes

can be applied to analyze DNA from diverse species of

bacteria; (iv) PCR products can be directly isolated

from the polyacrylamide gel and subsequently se-

quenced.

We suggested based on this study that there is at

least a similar power of discrimination between the

present gold-standard PFGE and a novel method,

ADSRRS fingerprinting. Although the ADSRRS fin-

gerprinting method may appear to be more complex

than RAPD technique, we found it to be fast and

reproducible.

The ADSRRS fingerprinting method described

here (with the same restriction enzymes, adapters

and primers) was successfully used also for epidemio-

logical studies of Acinetobacter baumannii and Ser-

ratia marcescens outbreaks (in preparation).

Numerous isolations of VREM in the Haematolog-

ical Unit of the Clinical Hospital in Gdansk in 1997 to

1999 indicated that the first nosocomial outbreak of

VREM had occurred in the country (Samet et al.,

1999; Hryniewicz et al., 1999; Kawalec et al., 2000).

Several lines of evidence obtained in this study of

large number of isolates suggested that the VanA

phenotype was selected by most likely one or two

independent events within the ward enterococcal pop-

ulation. This was supported by the observation of the

dominant existence of the two closely related

ADSRRS fingerprinting groups (A and B). Only

minor differences in ADSRRS fingerprinting patterns

observed in both groups revealed the ongoing evolu-

tionary diversification process within their populations

(no difference was observed using PFGE method,

group AB). The outbreak in the ward was polyclonal,

as demonstrated by the diversity of the distinguished

E. faecium ADSRRS fingerprinting patterns. The

observed relatedness of some of the E. faecium iso-

lates (groups A and B) suggested, however, that clonal

spread has also occurred. It may be postulated that

originally a single variant of the Tn1546-like trans-

poson was spread among the nonrelated enterococcal

strains circulating in the ward (Kawalec et al., 2000).

The present report confirmed the results described

previously, where VREM isolated between December

1996 and October 1997 were analyzed using PCR

fingerprinting (RAPD) technique (Samet et al., 1999).

The applied PCR fingerprinting system distinguished

two main closely related genotypes of the clinical

isolates from Haematological Unit (groups A and B,


and AB identified by ADSRRS fingerprinting and

PFGE method, respectively) and demonstrated only

small genetic heterogeneity among the isolates over

11 months.

It is not certain from our studies whether the VanA-

type resistance genes may have been transferred

between strains of E. faecium that were present on

the unit or whether two strains of VREM may have

been introduced into Haematological Unit independ-

ently over a short period of time, either by the transfer

from other hospitals or from the community. Data

presented here demonstrate the complexity of the

epidemiological situation concerning VREM that

may occur in a single medical ward.

Acknowledgements

We wish to thank A. PlCucienniczak and A. Masny

for their helpful suggestions during this work. This

work was supported by grant nos. 6 PO5A 151 21 and

DOT 67/2001 from The State Committee for Scientific

Research (Poland).

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Evaluation of a novel method based on amplification of DNA fragments surrounding rare restriction...

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