CTX-M-3 Type Beta-Lactamase Producing Shigella sonnei Isolates

Jpn. J. Infect. Dis., 61, 135-137, 2008
Short Communication
CTX-M-3 Type Beta-Lactamase Producing Shigella sonnei Isolates
from Pediatric Bacillary Dysentery Cases
Ziya Cibali Acikgoz, Ozgen Koseoglu Eser1* and Sesin Kocagöz2
Department of Microbiology, Faculty of Medicine, Fatih University, T.C. Saglık Bakanlıgı Ankara Ataturk
Education and Research Hospital; 1Department of Microbiology and Clinical Microbiology,
Faculty of Medicine, Hacettepe University, Ankara; and 2Department of Clinical Microbiology and
Infectious Diseases, Faculty of Medicine, Yeditepe University, Istanbul, Turkey
(Received September 14, 2007. Accepted December 12, 2007)
SUMMARY: In this study, 153 clinical isolates of Shigella were screened for extended-spectrum beta-lactamase
(ESBL) production by double-disk synergy test. After being confirmed by the combined disk-test and inhibitorcombined E-test strips, all positive isolates were tested for the bla gene by PCR and the enzymes by isoelectric
focusing. DNA sequencing of PCR products revealed that five Shigella sonnei isolates had CTX-M-3 type ESBL
enzymes. All of them were resistant to ampicillin, sulfamethoxazole and cefotaxime but susceptible to ofloxacin
and ceftazidime. All isolates displayed identical patterns in pulsed-field gel electrophoresis and enterobacterial
repetitive intergenic consensus PCR. This study reports multidrug-resistant (MDR) S. sonnei isolates producing
CTX-M-3 type ESBL from successive pediatric bacillary dysentery patients, indicating widespread and rapid
spread of CTX-M type ESBL in Shigella spp. To counter this emerging threat to public health, the surveillance
of CTX-M type beta-lactamases should be considered, together with measures designed to prevent outbreaks of
MDR Shigella in the community.
sequently administered (3).
The five isolates were further investigated by analytical
isoelectric focusing (IEF), polymerase chain reaction (PCR),
sequence analysis, enterobacterial repetitive intergenic consensus (ERIC)-PCR and pulsed-field gel electrophoresis
(PFGE). IEF was performed as described before (4). IEF
revealed that the isolates produced three beta-lactamases with
pIs of 7.0, 7.6 and 8.4. The latter was in accord with CTX-M
type ESBL. An IEF band of 7.0 might affirm a presence of
blaOXA gene, which was not specifically tested in the present
study. Although PCR amplification with blaSHV primers did
not confirm production of the remaining enzyme with a pI of
7.6, this may still be an SHV-type ESBL, as there are likely
various beta-lactamase genes yet to be discovered (5).
PCR was used to detect beta-lactamases of the SHV, TEM
and CTX-M families. PCR amplification methods using specific primers for blaCTX-M genes were performed in wholecell DNA of S. sonnei isolates. PCR amplification of blaCTX-M
group alleles (primers: blaCTX-M F 5´-TTT GCG ATG TGC
TGG CAT A-3´) (6) was found positive with an amplicon of
544 bp in all five isolates. PCR amplification of blaTEM (primers: F 5´-AAA CGC TGG TGA AAG TA-3´ and R 5´-AGC
GAT CTG TCT AT-3´) and blaSHV (primers: F 5´-ATG CGT
GCC AA-3´) group alleles was negative (7). The strains used
as PCR controls were E. coli ATCC 35218 and E. coli CT99
for blaTEM and E. coli SA1653 for blaSHV. A clinical CTX-Mpositive S. sonnei as a positive control and E. coli ATCC 25922
as a negative control were used for blaCTX-M. A further PCR
amplification using specific primers for blaCTX-M-3 (primers:
GCT TTC TGC CTT AGG TT-3´) was found to be positive
for the 780-bp amplicon size (8). Amplified PCR products of
bla genes were sequenced using an automated DNA sequencer
ABI Prism 310 (Applied Biosystems, Foster City, Calif., USA)
Extended-spectrum beta-lactamases (ESBLs) create significant therapeutic problems by inactivating almost all betalactams except cephamycins and carbapenems. Following the
detection of the first ESBL, SHV-1, in 1983, TEM-ESBLs
and SHV-ESBLs evolved greatly up to the late 1990s. Subsequently, a new epidemiological view of ESBLs has emerged,
with a dominant increase of CTX-M enzymes (1). Multidrugresistance in Shigella spp. is a growing concern all over the
world, and third-generation cephalosporins are used to treat
infections caused by multidrug-resistant (MDR) Shigella.
Here, we present successive community-acquired MDR
Shigella sonnei clinical isolates producing CTX-M-3 type
A total of 153 Shigella isolates were included in this study.
All were isolated in the Clinical Microbiology Laboratory of
Fatih University Hospital between 2003 and 2006. Specieslevel identification was performed by conventional biochemical methods plus specific antiserums. Species distribution was as follows: S. sonnei 122 (79.7%), S. flexneri 17
(11.1%), S. dysenteriae 13 (8.5%) and S. boydii 1 (0.7%).
At first, ESBL production of the isolates was investigated
synchronously with antibiograms by the double-disk synergy
test using cefotaxime, ceftazidime and amoxicillin-clavulanate
(AMC) disks (2). Five isolates evaluated as positive by this
method were also confirmed by clavulanic acid combination
disks for cefpodoxime, cefotaxime and ceftazidime, and
inhibitor-combined E-test strips (AB Biodisk, Solna, Sweden) containing ceftazidime/ceftazidime-clavulanate and
cefotaxime/cefotaxime-clavulanate. To interpret the results,
the CLSI ESBL phenotypic confirmatory tests for Escherichia
coli, Klebsiella pneumoniae and Proteus vulgaris were sub*Corresponding author: Mailing address: Department of Microbiology and Clinical Microbiology, Faculty of Medicine, Hacettepe
University, Sihhiye 06100, Ankara, Turkey. Tel: +90-312-3051560, Fax: +90-312-311-5250, E-mail: [email protected]
M) and Turkey (1 of 80 isolates, with CTX-M) (16-26). These
studies indicate that the most frequent species possessing
ESBL is S. sonnei. This may be due to the fact that S. sonnei is
the most frequently isolated species in industrialized countries.
The other studies before 2003 and the report from France in
2005 had no information of total isolate numbers or species
distribution. The study from Argentina (2005) included only
S. flexneri isolates. In the present study, 79.7% of all isolates
were S. sonnei.
Since all of our ESBL-producing isolates showed the same
genotype in ERIC-PCR and PFGE, it may be thought that
due to this case clustering, there might have been an outbreak. The route of transmission is unknown in this study. As
the route by which community infections arise is unclear in
ESBL infections, the population should fist be screened for
fecal carriage (14).
Shigellosis is endemic throughout the world. According to
the World Health Organization data, each year 1.1 million
people are estimated to die from shigellosis. Especially in
malnourished patients with shigellosis, antibiotic treatment
is recommended because it may resolve diarrhea, mitigate
symptoms and shorten the dysentery period. The first-line
drugs suggested by CLSI for shigellosis are ampicillin and
SXT (3). However, in case of infection by an MDR strain (i.e.,
one resistant to ampicillin, SXT and chloramphenicol), which
is not uncommon, second-line drugs are fluoroquinolones in
adults and third-generation cephalosporins in children. Thus,
in the near future, ESBL-producing Shigella spp. may present
a major challenge for dysentery treatment, especially in children, due to their third-generation cephalosporin resistance
and the probable side-effects of fluoroquinolones. Moreover,
fluoroquinolone-resistant Shigella strains are detected more
and more in various regions of the world (27).
The current low detection rate of ESBLs in Shigella isolates should not be misunderstood; the rising trend between
1999 and 2007 is adequate cause for alarm. Moreover, the
true proportion of ESBL-producing Shigella strains may be
higher than estimated. The drugs to be tested routinely for
intestinal Shigella and Salmonella isolates are ampicillin, SXT
and fluoroquinolones, with the third-generation cephalosporins and chloramphenicol tested only in the presence of an
extraintestinal isolate (3). Since extraintestinal infection due
to Shigella spp. is extremely rare, possible third-generation
cephalosporin resistance, which would signal the presence
of an ESBL, would be overlooked in routine antibiograms,
unless special screening studies are performed.
by Metis Biotechnology, Ankara, Turkey. Sequences were
compared with the GenBank sequence databases using the
BLAST suite of programs (9). DNA sequencing of the PCR
products revealed that the CTX-M-3 gene was positive for
all isolates (10). ERIC-PCR was performed using the ERIC2 primer (5´-AAG TAA GTG ACT GGG GTG ACG G-3´) as
previously described (11). For PFGE analysis, isolates were
prepared following the protocol of Brian et al., and the XbaI
(Boehringer, Mannheim, Germany) enzyme was used for digestion (12). By ERIC-PCR and PFGE, all the tested isolates gave
identical patterns based on Tenover’s criteria (13) (Figure 1).
The five strains were isolated from patients (3 females, 2
males), aged between 3 and 12 years, who were treated with
ciprofloxacin as outpatients. All were diagnosed as having
acute bacterial diarrhea at Fatih University Hospital between
2004 and 2005, and all fully recovered.
All five isolates were resistant to ampicillin, sulfamethoxazole (SXT), and cefotaxime but susceptible to ofloxacin
and ceftazidime in standard disk diffusion tests. They were
also susceptible to ceftazidime (MIC, 0.25 μg/ml) but resistant to cefotaxime (MIC, 256 μg/ml) by E-test. There were
>5 mm enlargements in cefotaxime and aztreonam inhibition zones next to the AMC disks but not in ceftazidime in
the double-disk synergy test. The combination disk diffusion
test was positive for cefotaxime but not for ceftazidime, with
a >5 mm diameter increase of the inhibition zone. In addition, cefpodoxime and cefpodoxime/clavulanic acid disks also
pointed to the presence of ESBL. Testing ESBL with E-test
strips gave identical results.
ESBLs elicit a serious global health concern because of
their resistance to a wide variety of currently available antimicrobials, which limits the therapeutic alternatives in many
critical infections. They are plasmid-mediated bacterial
enzymes that confer resistance to almost all beta-lactams
except for cephamycins and carbapenems. While the most
common enzyme types used to be SHV and TEM derivatives, CTX-M types are as widespread as them today. At
present, CTX-M type enzymes have over 50 members categorized in five groups, mainly in Enterobacteriaceae (14).
To the best of our knowledge, no ESBL-producing Shigella
strains were reported until a SHV-11 ESBL-producing S.
dysenteriae strain was found in India, in 1999 (15). Following this, several reports of ESBL-producing strains have been
published from different countries including France (1 strain
with SHV type enzyme), Korea (21 of more than 5,911 isolates; with TEM or CTX-M), Argentina (11 of more than
9,033 isolates; with CTX-M, TOHO, SHV or PER), Taiwan
(24 isolates of an outbreak; with CMY-2), Bangladesh (4 of
160 isolates, with ampC like or CTX-M), Israel (7 of 5,616
isolates, with CTX-M), Ireland (1 of 46 isolates with CTX-
We would like to thank Prof Z Gulay for providing the strains E. coli
CT99 and E. coli SA1653 for the study.
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