Download New variants of the tet(M) gene in Clostridium

Journal of Antimicrobial Chemotherapy (2006) 57, 1205–1209
doi:10.1093/jac/dkl105
Advance Access publication 24 March 2006
New variants of the tet(M) gene in Clostridium difficile clinical
isolates harbouring Tn916-like elements
Patrizia Spigaglia, Fabrizio Barbanti and Paola Mastrantonio*
Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanità,
Viale Regina Elena 299, 00161 Rome, Italy
Received 12 October 2005; returned 12 January 2006; revised 6 February 2006; accepted 7 March 2006
Objectives: To detect Tn916-like elements in Clostridium difficile clinical isolates from different time
periods and to analyse the genetic structure of these elements, in particular the tet(M) region.
Methods: Ninety C. difficile clinical isolates were examined by PCR assays for tet(M) and int, which are
markers for the Tn916 family of elements. Positive isolates were typed by PCR-ribotyping, and tetracycline
MIC values were evaluated by Etest. The genetic organization of the Tn916 elements was investigated by
PCR mapping and hybridization assays. The tet(M) region of eight selected C. difficile isolates was
sequenced.
Results: Nineteen isolates were tet(M)/int positive and the majority (12/19) belonged to PCR-ribotype R,
previously found to be predominant in clinical strains of more recent isolation. Eleven isolates were tetracycline resistant, three inducibly resistant and five susceptible. Fifty-eight per cent of the C. difficile
isolates harboured one Tn916 element and 42% harboured two. Most isolates showed elements with a
genetic organization very similar to that of Enterococcus faecalis DS16 Tn916. Sequence analysis highlighted variations in the leader peptide and six tet(M) variants were identified, five of which have never been
described before.
Conclusions: C. difficile isolates harbouring Tn916-like elements have mainly been isolated since 1997,
suggesting a recent circulation of these elements among C. difficile strains in Italian hospitals. Molecular
analysis of these Tn916-like elements showed that they may have different genetic structures and carry new
tet(M) alleles.
Keywords: tetracycline resistance, transposons, nosocomial pathogens
Introduction
Materials and methods
Clostridium difficile is the most common nosocomial pathogen
causing antibiotic-associated diseases.1,2 In this bacterium,
tetracycline resistance is commonly conferred by the tet(M)
gene, which is regulated by transcriptional attenuation.3 In
C. difficile 630, tet(M) was identified on the Tn5397 transposon, an element that differs from Tn916 in that it contains
a group II intron and has different integration/excision
modules.4 As elements related to Tn916 have recently been
found in some C. difficile clinical isolates,5 in this study, 90
C. difficile clinical isolates from different time periods were
examined for the presence and molecular characteristics of
Tn916-like elements.
C. difficile isolates and control strains
Ninety C. difficile strains isolated from patients in different
Italian hospitals, between 1986 and 2001, were examined in this
study. All isolates were tested for the presence of tet(M), int and
tndX genes. The last two genes were used as markers for Tn916- and
Tn5397-related elements, respectively.
Streptococcus pneumoniae PN20 strain was used as a control for the
int gene, whereas C. difficile 630 was used as a control for the tndX gene.
DNA extraction and detection of tet, int and tndX genes
Genomic DNA of each C. difficile strain was extracted using
a Nucleobond AXG20 Kit (Macherey-Nagel, Düren, Germany)
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*Corresponding author. Tel: +39-06-49902335; Fax: +39-06-49387112; E-mail: [email protected]
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Spigaglia et al.
Table 1. Primers used in this study for the characterization of tetracycline resistance elements
PCR target
Primers
tndX internal fragment
tndx1/tndx2
tet(M) internal fragment
int internal fragment
Tn916 from orf21 to tet(M)
TETMd/TETMr
INT1/INT2
CT3–CT4
CT9–CT10
CT15–CT16
CT17–CT18
CT21–CT22
CT25–CT26
CT27–CT28
CT27
TETMd
TETMr
M4
M5
M7
m11
tet(M) region
tet(K) internal fragment
TETKfor/TETKrev
tetA(P) internal fragment
TETAPfor/TETAPrev
tet(Q) internal fragment
TETQfor/TETQrev
tet(W) internal fragment
TETWfor/TETWrev
tet(32) internal fragment
TET(32)for/TET(32)rev
tet(36) internal fragment
TET(36)for/TET(36)rev
Reference or position on the sequence used for primer design
Tn5397 from C. difficile 630 (GenBank accession no. AF333235)
19041–19061/20648–20630
Tn916 from E. faecalis DS16 (GenBank accession no. U09422)
12521–12540/13600–13581
16682–16701/17606–17587
7
11507–11526
12521–12540
13600–13581
14018–13994
12014–12037
14070–14047
11803–11825
tet(K) from S. aureus (GenBank accession no. S67449)
187–204/355–338
tetA(P) from C. perfringens (GenBank accession no. L20800)
1415–1434/2090–2071
tet(Q) from Bacteroides thetaiotaomicron (GenBank accession no. X5871)
1301–1320/2204–2185
tet(W) from Clostridiaceae str. K10 (GenBank accession no. AY601650)
1681–1707/2138–2109
tet(32) from Clostridiaceae str. K10 (GenBank accession no. AJ295238)
783–800/1402–1383
tet(36) from Bacteroides sp. strain 139 (GenBank accession no. AJ514254)
2874–2893/3224–3205
from 10 mL of an overnight brain heart infusion (BHI)
culture.
The primers for tet, int and tndX gene amplification are
shown in Table 1. Amplifications for tet(M), int and tndX were
carried out as previously described.5,6 PCRs for the other tet gene
classes were performed using the following conditions: 5 min at
94 C, followed by 30 cycles of 94 C for 1 min, 50 or 55 C for
1 min and 72 C for 1 min.
Susceptibility testing and PCR-ribotyping
Tetracycline MIC values were determined by the Etest method
(AB Biodisk, Solna, Sweden). The breakpoint for tetracycline was
‡8 mg/L, according to the Clinical and Laboratory Standards
Institute (CLSI) criteria. Induction of resistance to tetracycline
was performed as previously described.5
PCR-ribotyping was performed with primers complementary to
conserved regions of the 30 end of the 16S rRNA gene and the 50 end
of the 23S rRNA gene, as previously described.6
Hybridization assays and molecular analysis of tetracycline
resistance determinants
as described in the DIG High Prime DNA labelling and detection
kit (Roche Diagnostics, Mannheim, Germany). The genomic DNA of
C. difficile isolates was digested with HindIII and transferred to a
nylon membrane (Roche Diagnostics) by Southern blotting.
Genetic organization of the Tn916-like elements was characterized by PCR mapping as previously described.5,7 Amplification of
the tet(M) region in eight selected isolates was carried out using the
set of primers reported in Table 1. PCR reactions were carried out in
a final volume of 50 mL with a reaction mixture containing buffer
(25 mM Tris–HCl/50 mM KCl/2.0 mM MgCl), 200 mM of each
deoxynucleoside triphosphate, 50 pmol of each primer and 2.5 U of
TakaRa ExTaqTM (Takara Shuzo Co., Ltd, Japan). Cycling parameters were denaturation at 95 C for 1 min, annealing at 50 C
for 1 min and extension at 72 C for 1 or 2 min (depending on
the size of the amplified fragments), followed by a final extension
step at 72 C for 5 min. PCR products obtained from the tet(M) region
were sequenced after purification by the NucleoSpin Extract kit
(Macherey-Nagel).
DNA sequencing and analysis
tet(M) and int PCR products were purified using the NucleoSpin
Extract kit (Macherey-Nagel), and DNA labelling was performed
Sequencing of the tet(M) region in the eight selected isolates was
carried out using the same primers designed for the PCR assay
(Table 1) and the Big Dye Terminator v.1.1 Cycle Sequencing
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tet(M) variants in Clostridium difficile
1
2
3
4
5
6
7
8
9
14.0 kb
8.7 kb
6.3 kb
Figure 1. Hybridization analysis of HindIII-digested DNA of C. difficile clinical
isolates with tet(M) and int probes. The signals obtained with both probes cobanded. The size of the hybridizing bands in kb is indicated on the left-hand side.
Lane 1, cd8; lane 2, digoxigenin-labelled l DNA HindIII-digested standard;
lane 3, cd12; lane 4, cd53; lane 5, cd11; lane 6, cd1911; lane 7, cd 60; lane 8,
cd63; lane 9, cd1920.
Kit (PE Biosystems, Foster City, CA, USA) on an Applied
Biosystems 3730 DNA Analyzer. Multiple sequence alignments
were performed using the European Bioinformatics Institute Clustal
W server, and the output was used for the construction of the phylogenetic tree by TreeView 1.4. The sequence similarity research
program was BLAST.
Nucleotide sequence accession numbers
The nucleotide sequences of the tet(M) region of C. difficile cd1911,
cd60, cd63, cd53, cd1920 and cd8 were submitted to the EMBL
database under accession numbers AJ973136, AJ973137,
AJ973138, AJ973139, AJ973140 and AJ973141, respectively.
Results and discussion
Nineteen of the 90 C. difficile isolates were positive for both
tet(M) and int (data not shown). None of these isolates was
positive for the tndX gene that was detected in 28% (25/90)
of the isolates. All 19 int positive isolates were collected
from 1997 to 2001. Twelve of the isolates (63%) belonged to
PCR-ribotype R, found to be predominant in clinical strains
isolated mainly in the years 2000 and 2001;5,6 the other seven
isolates belonged to different PCR-ribotypes. Eleven were resistant to tetracycline (MIC values between 8 and 32 mg/L), three
were inducibly resistant to tetracycline (MIC 4 mg/L before
induction and MICs between 8 and 12 mg/L after induction)
and five were susceptible (MICs between 0.047 and 4 mg/L)
(data not shown).
Hybridization assays were performed using the amplified products from tet(M) and int as probes. The signals obtained using
the two different probes co-banded (Figure 1). One isolate
showed a band at about 14 kb, four isolates at 8.7 kb and six
at 6.3 kb, indicating the presence of one element related to
Tn916. Eight strains showed two hybridizing bands at about
8.7 and 6.3 kb, respectively, suggesting that two Tn916-like
elements can co-exist in the same chromosome. No correlation
was found between the number of elements present in the
C. difficile isolates and the tetracycline resistance phenotype.
Eight of the 19 isolates examined had an element with a
genetic organization very similar to that of Enterococcus faecalis
DS16, whereas three isolates showed a different genetic organization. As already observed,5 sequence variations were located
in the DNA regions containing orf 16-15, orf 20-17 and orf 12tet(M). When two similar elements were in the same chromosome, PCR mapping could not be used for characterization. None
of the examined C. difficile isolates contained plasmids (data not
shown).
The tet(M) gene and the upstream region of eight selected
C. difficile strains (cd8, cd11, cd12, cd53, cd60, cd63, cd1911 and
cd1920) with different genotypic and phenotypic characteristics
were sequenced. Since different classes of the tet gene have been
found in clostridia (http://faculty.washington.edu/marilynr/), the
selected strains were also examined for tet(K), tet(Q), tet(W),
tetA(P), tet(32) and tet(36) by PCR; the results were negative
(data not shown).
Six different tet(M) variants, tet(M)1 to tet(M)6, were identified: cd11, cd12 and cd1911 harboured tet(M)1, cd60 tet(M)2,
cd63 tet(M)3, cd53 tet(M)4, cd1920 tet(M)5 and cd8 tet(M)6.
Sequence analysis revealed that except for tet(M)1, which
was 100% identical to the tet(M) gene found in Streptococcus
agalactiae 2603V/R (GenBank accession no. AE014233), the
other alleles did not show complete identity with other tet(M)
genes. The phylogenetic tree obtained by nucleotide sequence
comparison suggests that acquisition of these tetracycline resistance determinants from a hypothetical precursor carrying a
Tn916-like tet(M) gene is a relatively more recent event compared with the acquisition of the tet(M) gene by C. difficile 630
(Figure 2).
tet(M)1, 2, 3 and 4 variants were found in C. difficile isolates
resistant or inducibly resistant to tetracycline, and tet(M)5 and 6
were found in susceptible isolates. The organization of the element carrying tet(M)3, tet(M)6 and tet(M)1 of cd12 was similar
to that of E. faecalis DS16. This analysis was not possible in the
other two isolates harbouring tet(M)1 for the hypothesized presence of two Tn916-like elements in the chromosome. However,
the sequencing of the tet(M) region in these strains revealed that
there was no superimposition of different PCR products, suggesting that if two elements were present both elements carried the
same allele. tet(M)2, tet(M)4 and tet(M)5 were located on
elements that showed nucleotide variations compared with
E. faecalis DS16.
Analysis of the region upstream of the tet(M) genes showed
that tet(M)1, tet(M)3, tet(M)5 and tet(M)6 were preceded by a
complete leader peptide sequence, whereas this region was partially deleted in the region upstream of tet(M)2 and tet(M)4 for
the loss of 124 bp (data not shown). The residual transcriptional
attenuation activity could be maintained in these isolates since the
inverted repeats 7 (CCCTT) and 8 (TTCCC), forming the stemloop structure 7:8 in mRNA, a weaker terminator site, were still
present. The leader peptide DNA region of tet(M)3, 5 and 6 had
100% identity with that of Tn916 of E. faecalis DS16, whereas
that of tet(M)1 had 99% identity with one amino acid conservative mutation (M to I) in position four.
As the sequence of the region upstream of tet(M)5 and 6 does
not show variations compared with that of E. faecalis DS16
Tn916, the amino acid changes of Tet(M)5 and 6 proteins or
a post-transcriptional block may be responsible for the tetracycline susceptibility of the C. difficile strains carrying tet(M)5 and
tet(M)6.
No differences were observed in the –35 and –10 promoter
region of all tet(M) alleles compared with that of the Tn916 of
E. faecalis DS16, except for two nucleotide changes observed in
the –10 promoter region of tet(M)4, from TATTAT to TAATGT,
that could have some positive influence on transcription.
This study reports for the first time that one or two Tn916-like
elements can be present in C. difficile clinical isolates of recent
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Spigaglia et al.
Streptococcus mitis (AJ580977)
E. faecalis-Tn1545 (X04388)
Streptococcus cristatus (AY898750)
C. difficile cd1920-tet(M)5
S. pneumoniae (X90939)
C. difficile 630-Tn5397 (AF333235)
C. difficile cd53-tet(M)4
C. difficile cd8-tet(M)6
Neisseria gonorrhoeae (L12241)
Gardnerella vaginalis (U58985)
S. agalactiae (AE014233)
Neisseria meningitidis (X75073)
C. difficile cd1911-tet(M)1
Ureaplasma urealyticum (U08812)
S. aureus (M21136)
C. difficile cd63-tet(M)3
C. perfringens (AF329848)
C. difficile cd60-tet(M)2
E. faecalis (X56353)
Lactococcus lactis (DQ060148)
E. faecalis-Tn916 (U09422)
E. faecalis (X92947)
0.1
Figure 2. Unrooted neighbour-joining phylogenetic tree obtained from the nucleotide multiple alignment of the six C. difficile tet(M) variants identified in this study
and 16 other tet(M) gene sequences available in GenBank. The branch lengths are scaled in proportion to the extent of the change per position as indicated by the scale
bar. GenBank accession numbers are in parentheses.
isolation and that new tet(M) alleles are carried by these elements. Tn916 elements are known for their ability to transfer
antibiotic resistance genes and also virulence determinants
among bacteria belonging to different ecosystems.8–10 The
presence in C. difficile of elements related to Tn916 could
play an important role in the acquisition of new characteristics
that could increase the pathogenicity of this microorganism. For
this reason, further studies are necessary to monitor the spread of
these elements in strains circulating in hospital environments.
tet(36) gene amplifications. We are grateful to Tonino Sofia for
editing the manuscript. This work was partially supported by the
European Community’s Fifth Framework Programme ‘Quality of
Life and Management of Living Resources’, contract no.
QLK2-CT-2002-00843-ARTRADI.
Acknowledgements
References
We are indebted to Maria Del Grosso (Istituto Superiore di Sanità,
Rome, Italy) for supplying Streptococcus pneumoniae PN20, to
Anne Collignon (Université de Paris-Sud Châtenay-Malabry
Cedex, France) for supplying C. difficile 630 and to Karen Scott
(Rowett Research Institute, Bucksburn, Aberdeen, UK) for supplying control DNA for tet(K), tet(Q), tet(W), tetA(P), tet(32) and
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