752 Clinical Microbiology and Infection, Volume 10 Number 8, August 2004
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RESEARCH NOTE
Extended-spectrum b-lactamase (ESBL)
CTX-M-15-producing Escherichia coli and
Klebsiella pneumoniae in Sofia, Bulgaria
R. Markovska1, I. Schneider2,
E. Keuleyan3 and A. Bauernfeind2
1
Department of Microbiology, Medical University, Ministry of the Interior, Sofia, Bulgaria,
2
MICOER Institute, Munich, Germany and
3
Medical Institute, Ministry of the Interior, Sofia,
Bulgaria
ABSTRACT
During a survey of extended-spectrum b-lactamase (ESBL)-producing Enterobacteriaceae in
Bulgaria in 2001–2002, three isolates from Sofia
(two Escherichia coli, one Klebsiella pneumoniae)
showed cefotaxime MICs that were decreased in
the presence of clavulanate and were 2–8-fold
higher than those of ceftazidime. Resistance was
transferred to a sensitive recipient strain of E. coli.
Both wild-type and transconjugant strains produced a cefotaxime-hydrolysing b-lactamase of pI
8.8. Sequencing of the PCR product obtained with
oligonucleotide primers binding outside the
coding region identified this b-lactamase as
CTX-M-15. To our knowledge, this is the first
report of CTX-M-15 in Bulgaria.
Corresponding author and reprint requests: R. Markovska,
Department of Microbiology, Medical University, 2 Zdrave
Str., 1431 Sofia, Bulgaria
E-mail: markovska73@abv.bg
Keywords b-Lactamase, Bulgaria, CTX-M-15, ESBL,
Escherichia coli, Klebsiella pneumoniae
Original Submission: 21 December 2003; Revised
Submission: 18 March 2004; Accepted: 30 March 2004
Clin Microbiol Infect 2004; 10: 752–755
10.1111/j.1469-0691.2004.00929.x
CTX-M-type b-lactamases are a group of class A
extended-spectrum b-lactamases (ESBLs) that are
growing rapidly in importance [1,2]. CTX-M-1,
the first CTX-M-type b-lactamase, was detected in
an isolate of Escherichia coli from Germany in 1989
[2], and the overall number of cefotaximases has
now increased to c. 40 [3]. Most CTX-M-type
b-lactamases are 4–16-fold more active against
cefotaxime than against ceftazidime [1,4], but
enzymes with enhanced activity against ceftazidime have been detected recently, namely CTXM-15, -16, -19 and -27 [4–8]. CTX-M-15 was first
described in India in 2001 [5], and was then
identified subsequently in Poland, France, Turkey, Romania and the UK [4,9–12].
During a prospective survey of ESBLs in
Enterobacteriaceae from seven Bulgarian university hospitals in 2001–2002, 62 ESBL-producing
isolates were detected. Of these, three strains from
Sofia were identified (see below) as CTX-M-15
producers: E. coli Sof10 was isolated on 9 February
2001 from the urine of a female ambulatory
patient aged 60 years; Klebsiella pneumoniae Sof9
was isolated on 18 April 2002 from the blood
culture of a male patient, aged 47 years, in an
intensive care unit; and E. coli Sof11 was recovered
on 21 February 2002 from the blood culture of a
boy, aged 10 months, with pyelonephritis.
Antimicrobial susceptibilities were determined
by disk diffusion and an agar dilution procedure,
performed according to National Committee for
Clinical Laboratory Standards guidelines [13].
Plasmid transfer was performed on Mueller–
Hinton agar by mixing 0.8-mL portions of
18–24-h Luria–Bertani broth cultures of the donor
and recipient (E. coli K12:W3110 Rifr lac–) strains.
Transconjugants were selected on MacConkey
agar containing cefotaxime 2 mg ⁄ L and rifampicin 50 mg ⁄ L. Analytical isoelectric focusing was
done according to the method of Mathew et al.
[14] with modifications [2]. A bioassay was used
to determine the hydrolytic activity of the distinct
b-lactamase bands after isoelectric focusing as
2004 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 10, 749–772
Research Note 753
described previously [15]. Whole-cell DNA preparations were used as templates in specific PCRs
for the detection of CTX-M-type b-lactamase
genes. DNA was extracted with the GFX Genomic
DNA Purification Kit (Amersham Pharmacia
Biotech, Freiburg, Germany). Universal oligonucleotide primers CTX-M-UNI-V (5¢-CVATGTGCAGYACCAGTAA-3¢)
and
CTX-M-UNI-R
(5¢-ARGTGACCAGAAYMAGCGG-3¢), defined
from the nucleotide sequence of 18 blaCTX-M genes
including CTX-M-15, were used to amplify genes
coding for CTX-M-type b-lactamases (with an
annealing temperature of 61C). PCR amplification products from the transconjugant strains
were obtained with oligonucleotide primers P1c
(5¢-TCGTCTCTTCCAGAATAAGG-3¢) and P2c
(5¢-AAGGAGAACCAGGAACCACG-3¢), complementary to the flanking regions of the blaCTX-M
gene, and were subjected to automated sequencing
(ABI 3700; Applied Biosystems, Warrington, UK).
Whole-cell DNA preparations of the E. coli wildtype strains were typed by random amplified
polymorphic DNA analysis. Plasmid DNA of the
transconjugant strains was prepared with the
Qiagen Plasmid Midi Kit (Qiagen, Hilden, Germany) and subjected to plasmid fingerprint analysis by restriction with PstI.
Within the 14-month period from February
2001 to April 2002, three bacterial isolates from
three different medical centres in Sofia had a
phenotype suggestive of a CTX-M enzyme (diameter of inhibition zone for cefotaxime less than
that for ceftazidime) and gave positive results
with the National Committee for Clinical Laboratory Standards ESBL disk confirmatory method.
The cefotaxime resistance determinants, as well as
genes coding for resistance to tetracycline, gentamicin and tobramycin, were transferable by
conjugation from all three isolates to a sensitive
E. coli recipient. MICs for wild types, transconjugants and the E. coli recipient strain are shown in
Table 1. Each of the isolates had an individual
resistance profile. E. coli Sof10 displayed the
highest MICs and the broadest resistance pattern,
including resistance to ceftibuten. The MIC of
cefoxitin was not affected by the presence of an
AmpC b-lactamase inhibitor (Ro48-1220), indicating that impaired outer-membrane permeability
was a major resistance mechanism. All three wildtype isolates produced b-lactamases with pI
values of 5.4, 7.5 and 8.8, but cefotaxime was
hydrolysed only by the b-lactamase focusing at
pI 8.8.
With the use of CTX-M-type specific primers,
PCR products of the expected size of 585 bp
were obtained from all three isolates. Sequencing
of the entire coding region of blaCTX-M from the
transconjugant strains, followed by comparative
analysis with published sequences, revealed the
presence of CTX-M-15 in all three isolates. This
enzyme differs from CTX-M-3 by a single
amino-acid change (Asp-240-Gly) [5]. Random
amplified polymorphic DNA analysis of the two
E. coli wild-type isolates revealed no relatedness.
Plasmids isolated from transconjugant strains
showed identical PstI restriction profiles for the
two plasmids derived from E. coli Sof11 and
K. pneumoniae Sof9, while the plasmid derived
from E. coli Sof10 had a unique restriction
pattern.
Table 1. MICs (mg ⁄ L) for wild-type isolates and transconjugant strains producing CTX-M-15
Ceftazidime
+ Clavulanate
+ Sulbactam
Cefotaxime
+ Clavulanate
+ Sulbactam
Ceftibuten
Cefoxitin
+ Ro48-1220
Aztreonam
Imipenem
Ciprofloxacin
Tobramycin
Gentamicina
Amikacina
Tetracycline
Co-trimoxazole
Chloramphenicol
E. coli Sof10
R+ (E. coli Sof10)
E. coli Sof11
R+ (E. coli Sof11)
K. pneumoniae Sof9
R+ (K. pneumoniae Sof9)
E. coli R– RIFR
128
2
16
256
2
8
128
64
64
128
0.06
> 128
32
R
S
> 256
S
> 512
16
0.25
2
128
0.5
4
4
2
2
16
0.25
0.06
16
R
S
32
S
4
16
0.5
1
64
0.5
4
4
4
4
16
0.25
0.13
64
R
S
64
S
8
4
0.13
0,5
32
0.25
1
1
2
2
2
0.25
0.06
16
R
S
64
S
4
32
2
8
128
2
8
16
16
16
64
0.25
2
64
R
S
> 256
R
256
4
0.13
0.5
32
0.25
1
1
2
2
2
0.25
0.06
16
R
S
32
S
4
0.13
0.13
0.06
0.06
0.25
2
2
0.06
0.25
0.03
0.5
S
S
1
S
8
RIF, rifampicin; R+, transconjugants.
Results from disk diffusion test.
a
2004 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 10, 749–772
754 Clinical Microbiology and Infection, Volume 10 Number 8, August 2004
The identification of ESBL producers in Bulgaria dates back to 1992 [16]. Since then, the
incidence of ESBL-producing isolates has
increased to c. 5%, indicating that ESBLs represent an emerging problem in Bulgaria [17]. ESBLs
identified recently have included TEM-3, TEM-15,
SHV-5 and SHV-12 [18,19]. The present data
document the emergence of CTX-M-15-producing
isolates. Random amplified polymorphic DNA
typing demonstrated the genetic diversity of
the two E. coli isolates, which is consistent with
their different resistance patterns. K. pneumoniae
Sof9 and E. coli Sof11 had the same plasmid
fingerprint, which was distinct from that of E. coli
Sof10. These findings suggest the presence of two
different plasmids harbouring the blaCTX-M-15
gene.
Another 16 isolates (E. coli, K. pneumoniae,
Citrobacter freundii and Serratia marcescens) with
the CTX-M resistotype and producing a b-lactamase focusing at pI 8.8 have been identified
subsequently at different university centres in
Bulgaria (unpublished results). This development
may be related to the increasing use of oxyiminocephalosporins (e.g., ceftriaxone) in Bulgaria, with
the relative frequency of use of third-generation
cephalosporins increasing from 20% in 1999 to
50% in 2001 [20]. Several reports have suggested
that the ability of the ISEcp1 element to mobilise
and promote expression of CTX-M-15 is a significant factor in the rapid spread of the blaCTX-M-15
gene [4,5,10], and this could be the mechanism
underlying spread of the CTX-M-15 enzyme in
Bulgaria. Infections caused by bacteria producing
CTX-M-15 are often difficult to treat because of
multiresistance, and imipenem and amikacin
were the only antimicrobial agents found to be
active against the three isolates in the present
study. Further investigations are necessary to gain
a better understanding of the epidemiology and
genetic background of these enzymes, but the
present results demonstrate that CTX-M-15 has
emerged and spread in Bulgaria.
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RESEARCH NOTE
Emergence of CTX-M b-lactamaseproducing Enterobacteriaceae in Portugal:
report of an Escherichia coli isolate
harbouring blaCTX-M-14
E. Machado1, T. M. Coque2, R. Cantón2,
J. C. Sousa1 and L. Peixe1
1
Laboratório de Microbiologia, Faculdade de
Farmácia, Universidade do Porto, Porto, Portugal and 2Servicio de Microbiologı́a, Hospital
Universitario Ramón y Cajal, Madrid, Spain
ABSTRACT
CTX-M b-lactamases have been reported worldwide since their first description in the early
1990s, and are now endemic in some countries.
This report describes an isolate of Escherichia coli
producing CTX-M-14 that was recovered from the
faecal sample of a healthy subject in Portugal
without recent hospital or antibiotic exposure.
The presence of ISEcp1, 42 nucleotides upstream
of blaCTX-M-14, and its association with a 100-kb
conjugative plasmid, might result in wider dissemination of this enzyme in community and
hospital environments in Portugal.
Keywords b-Lactamase, CTX-M-14, Escherichia coli,
extended-spectrum b-lactamase, Portugal
Original Submission: 27 February 2004; Revised Submission: 23 March 2004; Accepted: 30 March 2004
Clin Microbiol Infect 2004; 10: 755–757
10.1111/j.1469-0691.2004.00930.x
Corresponding author and reprint requests: L. Peixe, Laboratório de Microbiologia, Faculdade de Farmácia, Universidade do Porto, Rua Anı́bal Cunha 164, 4050–047 Porto, Portugal
E-mail: lpeixe@ff.up.pt
The CTX-M b-lactamases are a growing group of
class A extended-spectrum b-lactamases that are
distinguished from TEM and SHV b-lactamases
by their much greater hydrolytic activity against
cefotaxime than against ceftazidime [1]. Members
of this family fit within six major groups (exemplified by CTX-M-1, CTX-M-2, CTX-M-8, CTX-M9, CTX-M-25 and Toho-2) on the basis of their
nucleotide and deduced amino-acid sequence
similarities [1]. Members of the different clusters
show differences of 10–30% in their amino-acid
sequences, while variants of the same cluster
show minor differences [1] (http://www.lahey.
org/studies/webt.htm). The blaCTX-M genes are
generally located on transferable plasmids of
variable size, and some have been shown to be
located within unusual class 1 integrons [1]. Two
different elements have been thought to play a
role in the mobilisation and expression of the
blaCTX-M genes: first, the ISEcp1 element, an
insertion sequence (IS) located upstream of many
blaCTX-M genes; and second, ORF513, a putative
transposase associated with unusual integrons
that might be involved in the acquisition of
different resistance genes. Finally, IS903 has been
found downstream of some blaCTX-M genes, suggesting a role in their dissemination [1–3].
The CTX-M enzymes, described first in the
early 1990s in South America and Europe, have
spread worldwide, and are now endemic in
South America, Japan, eastern Europe and Spain
[1,4–6]. In July 2003, a cefotaxime-resistant
isolate of Escherichia coli (EC-C498) was isolated
in Porto during a surveillance study of extended-spectrum b-lactamase-producing isolates in
northern Portugal. This isolate was recovered
from the faecal sample of a female, aged
76 years, without hospital or antibiotic exposure
in the 3-month period preceding collection of
the sample. MICs of b-lactams were determined
with Etests (AB Biodisk, Solna, Sweden), used
according to the procedures recommended by
the supplier. Susceptibility to non-b-lactam antimicrobial agents was determined by the disk
diffusion method according to National Committee for Clinical Laboratory Standards guidelines [7]. Characterisation of b-lactamases was
performed by isoelectric focusing, followed
by PCR amplification with primers to amplify
the blaTEM, blaSHV, blaCTX-M-9 and blaCTX-M-10
genes, and sequencing of the products obtained
[8].
2004 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 10, 749–772