Distribution of insertion sequences associated with Tn1546 and clonal diversity of vancomycin-resistant enterococci isolated from patients in Tehran, Iran.

Abstract

Infection with vancomycin-resistant enterococci (VRE) has caused a therapeutic problem. VanA and VanB resistant types are the predominant phenotypes among vancomycin resistant enetrococci. Transposon 1546 (Tn1546) harboring the vanA gene cluster, plays an important role in the horizontal transfer of vanA gene. In this study, we examined the phenotypic and genotypic diversity of a number of clinical VRE.
Twenty-four clinical VRE isolated from two university hospitals in Tehran were examined based on their antimicrobial susceptibility, Tn1546 related element organization and pulsed-field gel electrophoresis (PFGE) patterns. Integration of well-studied insertion sequence elements IS1216V, IS1542 and IS1251 was examined by PCR mapping and sequencing.
From 24 isolates, 15 isolates with VanA phenotype and 9 isolates with VanB phenotype were identified which both groups interestingly possessed the vanA gene. According to PCR mapping, our isolates were assigned to 6 main groups. In 14 (58.3%) isolates, IS1216V was inserted in vanX-vanY region and/or in truncated left-hand of Tn1546-like elements. In 11 (45.8%) isolates, both IS1216V and IS1542 were inserted in vanX-vanY and orf2-vanR regions, respectively and none of them harbored IS1251. Interestingly, PFGE of the isolates showed a high degree of diversity.
PCR mapping revealed that VanA elements in our isolates were highly heterogeneous. Overall, we found no correlation between transposon type and PFGE pattern. Genetic diversity of VRE provides practical information for epidemiological studies and our data showed horizontal transfer of VRE in this region.

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Page 1

Distribution of insertion sequences associated with Tn1546 and clonal
diversity of vancomycin-resistant enterococci isolated from patients in
Tehran, Iran
Oskoui M 1*, Farrokh P1
1Department of Bacteriology, Pasteur Institute of Iran, Tehran, Iran.
Received: December 2009, Accepted: February 2010.
ABSTRACT
Background and objectives: Infection with vancomycin-resistant enterococci (VRE) has caused a therapeutic problem.
VanA and VanB resistant types are the predominant phenotypes among vancomycin resistant enetrococci. Transposon 1546
(Tn1546) harboring the vanA gene cluster, plays an important role in the horizontal transfer of vanA gene. In this study, we
examined the phenotypic and genotypic diversity of a number of clinical VRE.
Materials and Methods: Twenty-four clinical VRE isolated from two university hospitals in Tehran were examined based on
their antimicrobial susceptibility, Tn1546 related element organization and pulsed-field gel electrophoresis (PFGE) patterns.
Integration of well-studied insertion sequence elements IS1216V, IS1542 and IS1251 was examined by PCR mapping and
sequencing.
Results: From 24 isolates, 15 isolates with VanA phenotype and 9 isolates with VanB phenotype were identified which both
groups interestingly possessed the vanA gene. According to PCR mapping, our isolates were assigned to 6 main groups. In
14 (58.3%) isolates, IS1216V was inserted in vanX-vanY region and/or in truncated left-hand of Tn1546-like elements. In
11 (45.8%) isolates, both IS1216V and IS1542 were inserted in vanX-vanY and orf2-vanR regions, respectively and none of
them harbored IS1251. Interestingly, PFGE of the isolates showed a high degree of diversity.
Conclusion: PCR mapping revealed that VanA elements in our isolates were highly heterogeneous. Overall, we found
no correlation between transposon type and PFGE pattern. Genetic diversity of VRE provides practical information for
epidemiological studies and our data showed horizontal transfer of VRE in this region.
Keywords: Vancomycin- resistant enterococci, Tn1546, insertion sequences, Iran.
INTRODUCTION
Enterococci are part of the normal flora in the
gastrointestinal tracts of humans (1, 2); however, they
can also be the main cause of nosocomial infection
especially in immunocompromised patients (3, 4).
Vancomycin-resistant enterococci (VRE) have presented
a global problem for treatment (3, 5, and 6). VRE are
phenotypically and genotypically heterogenous (7)
and there are six types of glycopeptide resistance in
enterococci (VanA to VanE and VanG) (8, 9). VanA
phenotype with acquired inducible resistance to both
vancomycin and teicoplanin, and VanB phenotype
with variable level of resistance to vancomycin but
susceptibility to teicoplanin are the most predominant
ones (10, 11). Recently, VRE with vanA genotype,
susceptible to teicoplanin (VanB phenotype-vanA
genotype), has become increasingly prevalent in Asia
(11).
VanA gene cluster is carried on transposon Tn1546
or closely related elements (5, 12) consisting of
vanR, vanS, vanH, vanA, vanX, vanY, vanZ genes
(3). orf1 and orf2 with transposition function are also
present in the left end of Tn1546. Due to integration
of insertion sequences (ISs), point mutation, and
deletions in nonessential genes or integration regions,
there are considerable variations among Tn1546 elements
(4, 5, 12).
Investigation of IS elements in VanA gene cluster
provides useful information for epidemiological
studies of the dissemination of VRE due to
horizontal transfer of Tn1546-like elements (3, 13).
The most common insertion sequences reported in
* Corresponding author: Mahvash Oskoui, Ph.D.
Address: Pasteur Institute of Iran. 69, Pasteur Ave.
Tehran-13164, Iran.
Tel: +98-21-66405535
Fax: +98-2166405535
E-mail: oskoui1@yahoo.com
Volume 2 Number 1 (March 2010) 14-21
14

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DIVERSITY OF VANCOMYCIN RESISTANT ENTEROCOCCI IN TEHRAN
vanA gene cluster are IS1216V, IS1542, IS1251,
and IS1476 (14). Although IS1216V is known in
most of the vanA elements, the other IS elements
appear to be geographically restricted (14). Beside
horizontal transfer of resistance gene cluster, clonal
dissemination of VRE was determined in various
studies (14, 15).
Although vancomycin-resistant enterococci have
Reference Amplicon size (bp) Sequences(5’ to 3’)Primer
271119
ACGTTAAGAAAGTTTTAGTGG
GCCCTTTTAGGAATGG
orf 1AF
orf 1 AR
27 1696
CATACATGCGCCATTGAGATA
GTTAGTCCATCCTCGCTTGAT
orf 1 BF
orf 1 BR
14
26
Variable
AGGATTATATAAGAAAACCCG
GTTAGTCCATCCTCGCTTGAT
IS1216V R
orf 1 BR
26540
TTGCGGAAAATCGGTTATATTC
AGCCCTAGATACATTAGTAATT
orf 2 F
orf 2 R
14
26
Variable
AGGATTATATAAGAAAACCCG
GCCCTAGATACATTAGTAATT
IS1216V R
orf 2 R
25
26
Variable
GAATCGCTTTTACTGCTTCTC
CGGATTATCAATGGTGTCGTT
IS1542 F
vanR R
26 Variable
TTGCGGAAAATCGGTTATATTC
TTCTAAAGCTGCCATATTGC
orf2 F
IS1542 R
261485
TTGCGGAAAATCGGTTATATTC
CGGATTATCAATGGT GTC GTT
orf2 F
vanR R
212338
TTGGTTATAAAATTGAAAAATAA
TCCTTTCAAAATCCAAACAGTTT
vanS F
vanH R
21
3
Variable
TTGGTTATAAAATTGAAAAATAA
AGGATTATATAAGAAAACCCG
vanS F
IS1251 R
3Variable
TCAGACGACCTTGAGAAC
TCCTTTCAAAATCCAAACAGTTT
IS1251 F
vanH R
261029
ATGAATAGAATAAAAGTTGCAATAC
CCCCTTTAACGCTAATACGAT
vanA F
vanA R
211947
ATGGAAATAGGATTTACTTT
TTACCTCCTTGAATTAGTAT
vanX F
vanY R
21
14
Variable
ATGGAAATAGGATTTACTTT
AGGATTATATAAGAAAACCCG
vanX F
IS1216V F
14
21
Variable
ACCTTCACGATAGCTAAGGTT
TTACCTCCTTGAATTAGTAT
IS1216V R
vanY R
21 454
TTATCTAGAGGATTGCTAGC
AATGGGTACGGTAAACGAGC
vanZ F
vanZ R
7 433
CAAAGCTCCGCAGCTTGCATG
TGCATCCAAGCACCCGATATAC
vanB F
vanB R
Table 1. Nucleotide sequences of PCR primers used in this study.

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16
OSKOUI ET AL . IRAN. J. MICROBIOL. 2 (1) : 14-21
been reported worldwide, investigation of resistant
isolates from different geographic locations
provides useful information (16, 17). In this study,
our goal is determination of the phenotypic as
well as genotypic diversity of clinical VRE in this
region with using antimicrobial susceptibility, PCR
mapping and Pulse-field gel electerophoresis.
MATERIALS AND METHODS
Bacterial isolates and identification. A total of 24
VRE clinical isolates, collected between May 2006 and
May 2007 from Milad and Sina University Hospitals
in Tehran, were studied. These VRE were isolated
from nearly 500 enterococci. With the exception of
isolates from blood samples, all enterococci were
isolated from urine specimens. These isolates were
identified by conventional biochemical reactions
(18). E. faecium BM4147, E. faecalis ATCC 29212,
and E. faecalis V583 were used as control strains.
Antimicrobial agents and MIC determination.
Antimicrobial susceptibility of the isolates were
tested by the disc diffusion method and interpreted
according to the Kerby-Bauer method (19). The
antibiotics (MAST Diagnostics Ltd. Merseyside,
England) used for disc diffusion assays included
vancomycin (30μg), teicoplanin (30μg), tetracycline
(30μg), erythromycin (15μg), chloramphenicol
(30μg), ampicillin (10μg), and ciprofloxacin (5μg).
Minimum inhibitory concentration (MIC) of
vancomycin (SERVA FEINBIOCHEMICA GmbH
& Co., Germany) and teicoplanin were determined
using broth microdilution (19) and Etest (AB Biodisk,
Solna, Sweden), respectively.
Characterization of Tn1546-like elements.
Extraction of chromosomal and plasmid DNA were
Table 2. The phenotypic and genetic characteristics of vancomycin resistant isolates of enterococci isolated from patients in Tehran.
‡ Vm, Vancomycin; Tc, teicoplanin; Am, ampicillin; Cm, chloramphenicol; Cp, ciprofloxacin; Em, erythromycin; Te,
tetracycline
Isolate Source
MIC (μg/ml)Van†
Resistance profile‡
Transposon
Type
Vancomycin TeicoplaninPhenotypeGenotype
E. faecium
B-201 IPI
495 IPI
105 IPI
107 IPI
431 IPI
B-364 IPI
N-117 IPI
557 IPI
390 IPI
110 IPI
436 IPI
108 IPI
B-269 IPI
102 IPI
B-148 IPI
106 IPI
109 IPI
E-83 IPI
urine
urine
urine
urine
urine
urine
urine
urine
urine
urine
urine
urine
urine
urine
blood
urine
urine
urine
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
128
128
96
64
64
64
64
64
64
64
64
64
48
32
1.5
1
0.5
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
B
B
B
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
Vm, Tc, Cp, Em, Te
Vm, Tc, Cp, Em, Te, Am, Cm
Vm, Tc, Cp, Em, Te, Am, Cm
Vm, Tc, Cp, Em, Am, Cm
Vm, Tc, Cp, Em, Te, Am, Cm
Vm, Tc, Cp, Em, Te
Vm, Tc, Cp, Em, Te, Am
Vm, Tc, Cp, Em, Te, Am, Cm
Vm, Tc, Cp, Em, Te, Am, Cm
Vm, Tc, Cp, Em, Am
Vm, Tc, Cp, Em, Te, Am, Cm
Vm, Tc, Cp, Em, Am, Cm
Vm, Tc, Cp, Em
Vm, Tc, Cp, Em, Te, Am
Vm, Tc, Cp, Em, Te, Am
Vm, Cp, Em, Te, Am, Cm
Vm, Cp, Em, Te, Am
Vm, Cp, Em, Te
A1
B3
A1
B1
F7
F6
C
F1
B2
B1
E1
B1
E2
A3
A2
B1
D
F5
E. faecalis
523 IPI
N-89 IPI
E-125 IPI
578 IPI
524 IPI
B-219 IPI
urine
urine
urine
urine
urine
urine
>256
>256
128
32
16
16
1B
B
B
B
B
B
A
A
A
A
A
A
Vm, Cp, Em, Te, Am
Vm, Cp, Em, Te, Cm
Vm, Cp, Em, Te
Vm, Cp, Em
Vm, Cp, Em, Te, Am
Vm, Cp, Em, Te
F3
F2
F8
F4
F10
F9
0.5
0.5
0.38
1.5
1.5

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DIVERSITY OF VANCOMYCIN RESISTANT ENTEROCOCCI IN TEHRAN
performed using Bacterial Genomic and Plasmid
Miniprep kits (Metabion, Martinsried, Germany).
VanA and VanB-type enterococci were examined by
PCR with primers specific for the vanA and vanB
genes.
For structural analysis of Tn1546-like elements,
PCR was carried out with previously published
primers for orf1, orf 2, orf 2-vanR, vanS-vanH, vanA,
vanX-vanY, and vanZ (Table 1). Presence of well-
studied IS elements IS1216V, IS1542 and IS1251 in
the orf 2-vanR , vanS-vanH and vanX-vanY intergenic
regions were confirmed with additional PCR by one
primer in the published sequence of Tn1546 and one
in the published IS sequence (Table 1). In order to
determine precise left ends of the Tn1546, DNA
fragments were amplified with a combination of
Tn1546-derived primers and primers based on the
IS1216V which was inserted at the left end of the
transposon. The primer sequences and amplicon size
of products are listed in Table 1.
PCR amplification was carried out on a Eppendorf
thermal cycler with the following protocol: initial
denaturation at 95°C for 4 min; this was followed by
30 cycle of DNA denaturation at 95°C for 30 S, primer
annealing at 55-56°C for 1 min and DNA extension at
72°C for 1 min; and final extension at 72°C for 10 min.
DNA sequence analysis. PCR amplicons of
vanX-vanY and orf2-vanR regions which were
larger than those of the prototype vanA gene cluster
were sequenced with IS1216V and IS1542 primers.
To determine the DNA sequence of the left end of
the VanA transposon derivatives, PCR products of
this region were sequenced with IS1216V primers
(Macrogen Research, Seoul, Korea).
Transposon
Type
PCR amplification of Tn1546-like elements
vanZ
vanXF
IS1216VF
IS1216VR
vanYR
vanXF
vanYR
vanA
vanSF
vanHR
orf2F
vanRR
orf2F
IS1542R
IS1542F
vanRR
S1216VR
orf2R
orf2
IS1216VR
orf1BR
orf1B orf1A
No. of
isolates
A1
+++++++++++ ----+----*
++2
A2
+++++++-+-----+----++1
A3
+++++++-++-+---- --1
B1
+++++++++++---- +++-4
B2
+-+++++-++----+++-1
B3
++--+--+-----+++-1
C+---++-+- ----+++-1
D+++-++----+------1
E1
+--++++------+++-1
E2
+---+-+--++------1
F1
+---++-------+----++1
F2
+---+-+------+---- ++1
F3
----+-------- +----++1
F4
---++-+---+------1
F5
----+-+---+---- --1
F6
+--++-----+------1
F7
----++----+---- --1
F8
+---+-----+------1
F9
----+-----+------1
F10
+---+---------------1
Table 3. +, amplicons the same size to prototype, ++, amplicons larger than prototype and -, absence of an amplicon with
particular primers.* PCR reaction was not performed.

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OSKOUI ET AL . IRAN. J. MICROBIOL. 2 (1) : 14-21
Pulse-field gel electerophoresis (PFGE). PFGE
was performed as described previously (20). Genomic
DNA was digested with SmaI (Fermentas, Vilnius,
Lithuania), and separated on a 1% agarose gel using
a contour-clamped homogeneous-field apparatus
(CHEF DR III system; Bio-Rad Laboratories,
Richmond, CA). Salmonella Braenderup H9812
was used as molecular weight marker after XbaI
(Fermentas, Vilnius, Lithuania) digestion. The
agarose gels were run at 14°C and 6 V/cm for 21 h,
with a linear pulse time of 5 to 40 sec at an angle
of 120 degrees. The banding patterns were analyzed
using Gelcompar II version 4.0 (Applied Maths Sint-
Matens-Latem, Belgium).
RESULTS
Bacterial isolates and antibiotic resistance.
Eighteen Out of 24 VRE were E. faecium and
the remaining were E. faecalis. All the isolates
were resistant to vancomycin, ciprofloxacin and
erythromycin discs. Furthermore, 79.2%, 66.7%,
62.5% and 41.7% of the isolates were resistant
to tetracycline, ampicillin,
chloramphenicol, respectively (Table 2). The MIC of
teicoplanin and
vancomycin and teicoplanin of the isolates are given
in Table 2. Fifteen out of 18 E. faecium showed the
VanA phenotype, while all the E. faecalis and 3 of the
E. faecium displayed VanB phenotype.
Structural analysis of Tn1546 element by PCR
mapping. When PCR was carried out with the vanA
specific primer, PCR product of expected size was
obtained from 15 isolates with VanA phenotype. None
of the 9 isolates with VanB phenotype possessed the
vanB gene, but all of them harbored the vanA gene.
PCR mapping of Tn1546-like elements of 24
isolates revealed 6 main different transposon types
(A-F) according to the patterns of ISs inserted into
Tn1546 (Table 3). Type A was characterized by an
IS1216V insertion in the vanX-vanY intergenic region
and an IS1542 insertion in the orf2-vanR region.
Type B was specified by the presence of two copies
of IS1216V at the left-hand of Tn1546 and the vanX-
vanY region and an IS1542 insertion in the orf2-
vanR intergenic region. Type C was indicated with
one copy of IS1216V in the left-hand of Tn1546 and
the presence of IS1542 in the orf2-vanR intergenic
region. Type D and E were characterized with one
copy of IS1216V in the vanX-vanY region and one
copy in the left-hand of Tn1546, respectively. Group
Fig. 1. PFGE analysis of vancomycin resistant E. faecium (A) and E. faecalis (B). The phylogenetic tree is based on cluster
analysis of the unweighted pair group method with average linkages (UPGAMA) and Dice analysis.

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DIVERSITY OF VANCOMYCIN RESISTANT ENTEROCOCCI IN TEHRAN
F had no insertion sequences (Table 3).
Through amplification of intergenic regions, only
9 and 6 isolates showed larger size of amplicon,
approximately 3000bp, in the vanX-vanY and orf
2-vanR regions, respectively (Table 3).
Sequence analysis of the VanA transposons.
Sequencing of vanX-vanY and orf2-vanR amplicons
larger than those of the prototype VanA gene cluster
(larger than 3000bp) showed that IS1216V and
IS1542 were inserted in these regions, respectively.
We published partial sequence of IS1216V and
IS1542 with access numbers of FJ416860, FJ416861,
GQ273971 and GQ273972 in GenBank (www.ncbi.
nlm.nlh.gov/Genbank/submit.html). Sequencing of
amplicons of the left end of Tn1546-like elements
confirmed insertion of IS1216V in this region.
PFGE profiles. Analysis of the 18 E. faecium and
6 E. faecalis banding patterns differentiated 9 and 5
PFGE t
ypes respectively, with a similarity value of 0.7
(fig.1). So, the VRE isolates showed a high degree
of heterogeneity. Among vancomycin resistant E.
faecium, 9 isolates, with more than 78% similarity in
their banding patterns, were the prevalent type.
DISCUSSION
Up to now, phenotypic and genotypic varieties
of the vancomycin-resistant enterococci have been
investigated in several previous studies (13, 21).
In the present study, we compared the phenotypic
(antibiotic resistant patterns) as well as genotypic
traits of 24 VRE, isolated from patients admitted
in two major hospitals of Tehran. The predominant
species in this study were E. faecium (75%) with E.
faecalis accounting for 25% of the remaining isolates.
Overall, the VRE isolates in spite of resistant to
vancomycin, were 100% resistant to erythromycin and
ciprofloxacin; furthermore, ampicillin showed a high
frequency of resistance among E. faecium (94.5%).
Similar to the previous study (22), chloramphenicol
was the only drug that showed a lower rate of
resistance among our isolates. Since consumption of
antibiotics and the related selective pressures causes
antibiotic resistance in community, it seems that high
level of resistance to these antibiotics is related to their
high consumption (1).
All the VRE harbored the vanA gene; however, 9
(37.5%) of them exhibited the VanB phenotype-vanA
genotype. Incongruence of the VanB phenotype-vanA
genotype has been found in Japan, Taiwan, and Korea
(23), but to our knowledge, this is the first report of
VanB-vanA VRE from humans in Iran.
According to PCR mapping, VanA elements
were highly heterogeneous and none of them were
completely similar to the prototype BM4147. Similar
results were also obtained with previous studies (13, 14).
Based on PCR and sequencing results, IS1216V was
found in 14 isolates (58.3%) in vanX-vanY and/or in
the left-hand of Tn1546-like elements and had higher
frequency among VRE. In 11 isolates (45.8%), both
IS1216V and IS1542 were inserted in vanX-vanY and
orf 2-vanR regions. IS1251 was identified neither with
vanS F-vanH R primers nor with published primers of
IS1251 and vanS/vanH through PCR reaction. Thus,
the distribution of ISs in Tn1546-like elements of our
isolates is similar to those among Europeans and
Koreans (2, 21, 24) but not to American VRE isolates
(1, 12, 13).
vanZ, as a nonessential gene in Tn1546, was
detected in 19 (79.2%) of our isolates using PCR. The
vanR, vanS, vanH and vanX genes are essential for
the expression of vanA resistance, therefore, it was
surprising that some isolates lacked these amplicons
after PCR with intergenic region primers. PCR results
obtained from each single gene showed that in 100%,
87.5%, 79.2% and 62.5% of the isolates vanR, vanS,
vanH and vanX were amplified, respectively (data
was not shown). In this study, negative PCR reactions
were repeated several times, but absence of PCR
products may indicate disruptions or insertions in
these regions and these kinds of difficulties with
amplifying these regions of some VanA elements
have been reported previously (25).
Many of the studied VanA elements (18 isolates)
lacked one or more amplicons in the left-hand of
Tn1546, corresponding to genes orf 1 and orf 2
associated with transposition functions. Similar
findings were reported in Europe and Korea (14, 25).
Based on some reports, point mutation, insertion of
IS elements and deletion of intergenic region can be
responsible for the VanB phenotype-vanA genotype
(2, 11, 23). It seems that loss of some Tn1546 parts
can be responsible for this change in our isolates.
Using PFGE we found high degree of diversity
among the isolates. While 6 E. faecium with more
than 78% similarity had both IS1216V and IS1542
in vanX-vanY and orf 2-vanR regions, we generally

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OSKOUI ET AL . IRAN. J. MICROBIOL. 2 (1) : 14-21
found no correlation between the positions of Insertion
sequences in the Tn1546-like elements and the isolates
PFGE types. The genetic diversity among Tn1546
elements was shown in several previous studies (14,
24, and 25). The variability in transposon type in
various countries may be explained by the different
antibiotic selective pressures against glycopeptides
(2, 25), as well as the geographic differences in the
transposon distribution (14) and movement of ISs in
Tn1546 during outbreak (25).
In conclusion, our results revealed high degree of
diversity and unique characterization among VRE in
clinical specimens in this region. Thus, the genetic
diversity indicated horizontal transfer of VRE rather
than their clonal dissemination in this region and
offered useful information for further epidemiological
studies.
Acknowledgment. We are grateful to Dr.
Mohammad Mehdi Feizabadi for providing the control
isolates used in this study and also we thank Dr. Ali
Nojoumi for carefully reading of the manuscript.
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